Stem Cells

Neuron 

Oligodendrocyte 

Neural 

progenitor 

cell 

Astrocyte 

FOCUS 

Stem Cells

focus 

Stem Cells 

EDITORS 

Barbara Pauly 

Senior Editor 

pauly@embo.org | T +49 6221 8891 109 

Barbara joined EMBO Reports in September 2008. She completed her PhD at the University 

of Munich, focusing on signal transduction in the fresh water polyp Hydra. She worked at the 

University of California at Berkeley as a post-doctoral researcher, studying the role of the actin 

cytoskeleton in endocytosis in mammalian cells. 

Daniel Klimmeck 

Editor 

d.klimmeck@embojournal.org | T +49 6221 8891 407 

Daniel received his PhD in 2008 with work on ion channel signaling in sensory neurons in the 

laboratory of Stephan Frings at the University of Heidelberg. As a postdoc, he focused on the 

molecular characterization of cancer and hematopoietic stem cells with Andreas Trumpp at 

the DKFZ and Jeroen Krijgsveld at EMBL. Daniel joined The EMBO Journal in 2015. 

Celine Carret 

Editor 

c.carret@embomolmed.org | T +49 6221 8891 411 

EMBO 

Molecular 

Medicine 

Céline Carret completed her PhD at the University of Montpellier, France, characterising host 

immunodominant antigens to fight babesiosis, a parasitic disease caused by a unicellular 

Apicomplexan parasite closely related to the malaria agent Plasmodium. She further 

developed her post-doctoral career on malaria working at the Wellcome Trust Sanger Institute 

in Cambridge, UK and Instituto de Medicina Molecular in Lisbon, Portugal. Céline joined EMBO 

Molecular Medicine as a Scientific Editor in March 2011. 

Maria Polychronidou 

Editor 

msb@embo.org | T +49 6221 8891 410 

Maria received her PhD from the University of Heidelberg, where she studied the role of 

nuclear membrane proteins in development and aging. During her post-doctoral work, she 

focused on the analysis of tissue-specific regulatory functions of Hox transcription factors 

using a combination of computational and genome-wide methods. 

emboj.embopress.org | embor.embopress.org | embomolmed.embopress.org | msb.embopress.org

UPCOMING MEETINGS 

5th May 

22nd May 

3rd June 

EMBO workshop: Neural control of metabolism and eating 

behaviour, Portugal 

THE STEM CELL NICHE - DEVELOPMENT & DISEASE, 

Copenhagen, Denmark 

EMBO | EMBL Symposium: Hematopoietic Stem Cells: from the 

embryo to the aging organism, Heidelberg, Germany 

Céline Carret (EMBO Molecular Medicine) 

Barbara Pauly (EMBO Reports) 

Barbara Pauly (EMBO Reports) and Daniel 

Klimmeck (The EMBO Journal) 

6th June EMBL partnership: Translational medicine, Germany Céline Carret (EMBO Molecular Medicine) 

12th June 

GRC conference: Membrane Transporters: Translating 

Molecules to Medicine, Lucca, Italy 

Barbara Pauly (EMBO Reports) 

22nd June ISSCR 2016 Annual Meeting, USA Daniel Klimmeck (The EMBO Journal) 

26th June EMBO/EMBL symposia: Innate Immunity, Germany Céline Carret (EMBO Molecular Medicine) 

18th July Synthetic Biology: Engineering, Evolution & Design, USA Maria Polychronidou (Molecular Systems Biology) 

7th August 

16th August 

GRC: Tissue Niches & Resident Stem Cells in Adult Epithelia, 

Hong Kong, PRC 

EMBO Workshop: Molecular Mechanisms of Ageing and 

Regeneration, GR 

Daniel Klimmeck (The EMBO Journal) 

Daniel Klimmeck (The EMBO Journal) 

27th August EMBL Conference: Transcription and Chromatin, Germany Maria Polychronidou (Molecular Systems Biology) 

12th September Annual German Stem Cell Network Conference, GER Daniel Klimmeck (The EMBO Journal) 

14th September 

EMBL Conference: Proteomics in Cell Biology and Disease 

Mechanisms, Germany 

Maria Polychronidou (Molecular Systems Biology) 

18th September Behr Symposium: Stem Cells and Cancer, Heidelberg Barbara Pauly (EMBO Reports) and Daniel 

Klimmeck (The EMBO Journal) 

13th October 9th Berling Summer Meeting, Germany Maria Polychronidou (Molecular Systems Biology) 

26th October Keystone: Translational vaccinology for global health, UK Céline Carret (EMBO Molecular Medicine) 

11th December Cell Symposium, Hallmarks of Cancer, BE Daniel Klimmeck (The EMBO Journa 

or other meetings attended by editors, please see http://t.co/Oxv86K96Ct

CONTENTS 

Full Articles 

EMBO Reports 

Yap1 is dispensable for self-renewal but required for proper 

differentiation of mouse embryonic stem (ES) cells 

HaeWon Chung, Bum-Kyu Lee, Nadima Uprety, Wenwen Shen, Jiwoon Lee, Jonghwan Kim 

EMBO Reports Published online 25.02.2016 

DOI:10.15252/embr.201540933 

Resistance of glia-like central and peripheral neural stem 

cells to genetically induced mitochondrial dysfunction — 

differential effects on neurogenesis 

Blanca Díaz-Castro, Ricardo Pardal, Paula García-Flores, Verónica Sobrino, Rocío Durán, 

José I Piruat, José López-Barneo 


DOI:10.15252/embr.201540982 

UTX inhibits EMT-induced breast CSC properties by 

epigenetic repression of EMT genes in cooperation with 

LSD1 and HDAC1 

Hee-Joo Choi, Ji-Hye Park, Mikyung Park, Hee-Young Won, Hyeong-seok Joo, Chang Hoon 

Lee, Jeong-Yeon Lee, Gu Kong 


DOI:10.15252/embr.201540244 

Chromatin remodeling and bivalent histone modifications 

in embryonic stem cells 

Arigela Harikumar, Eran Meshorer 


DOI:10.15252/embr.201541011 

continued overleaf

CONTENTS 

One-page highlights 

EMBO Reports 

Integrative genomics positions MKRN1 as a novel 

ribonucleoprotein within the embryonic stem cell gene 

regulatory network 

Paul A Cassar, Richard L Carpenedo, Payman Samavarchi-Tehrani, Jonathan B Olsen, 

Chang Jun Park, Wing Y Chang, Zhaoyi Chen, Chandarong Choey, Sean Delaney, Huishan 

Guo, Hongbo Guo, R Matthew Tanner, Theodore J Perkins, Scott A Tenenbaum, Andrew 

Emili, Jeffrey L Wrana, Derrick Gibbings, William L Stanford 


DOI:10.15252/embr.201540974; 

Selective influence of Sox2 on POU transcription factor 

binding in embryonic and neural stem cells 

Tapan Kumar Mistri, Arun George Devasia, Lee Thean Chu, Wei Ping Ng, Florian Halbritter, 

Douglas Colby, Ben Martynoga, Simon R Tomlinson, Ian Chambers, Paul Robson, Thorsten 

Wohland 


DOI:10.15252/embr.201540467; 

Mitochondrial metabolism in hematopoietic stem cells 

requires functional FOXO3 

Pauline Rimmelé, Raymond Liang, Carolina L Bigarella, Fatih Kocabas, Jingjing Xie, 

Madhavika N Serasinghe, Jerry Chipuk, Hesham Sadek, Cheng Cheng Zhang, Saghi 

Ghaffari 


DOI:10.15252/embr.201439704; 

DAZL regulates Tet1 translation in murine embryonic 

stem cells 

Maaike Welling, Hsu-Hsin Chen, Javier Muñoz, Michael U Musheev, Lennart Kester, 

Jan Philipp Junker, Nikolai Mischerikow, Mandana Arbab, Ewart Kuijk, Lev Silberstein, 

Peter V Kharchenko, Mieke Geens, Christof Niehrs, Hilde van de Velde, Alexander van 

Oudenaarden, Albert JR Heck, Niels Geijsen 


DOI:10.15252/embr.201540538 

Distinct germline progenitor subsets defined through 

Tsc2–mTORC1 signaling 

Robin M Hobbs, Hue M La, Juho-Antti Mäkelä, Toshiyuki Kobayashi, Tetsuo Noda, Pier 

Paolo Pandolfi 


DOI:10.15252/embr.201439379 

continued overleaf

CONTENTS 

One-page highlights continued 

EMBO Reports 

Epigenetic predisposition to reprogramming fates in 

somatic cells 

Maayan Pour, Inbar Pilzer, Roni Rosner, Zachary D Smith, Alexander Meissner, Iftach 

Nachman 


DOI:10.15252/embr.201439264; 

Harnessing the apoptotic programs in cancer stem-like cells 

Ying-Hua Wang, David T Scadden 


DOI:10.15252/embr.201439675 

Effects of inflammation on stem cells: together they strive? 

Caghan Kizil, Nikos Kyritsis, Michael Brand 


DOI:10.15252/embr.201439702;

Scientific Report 

Yap1 is dispensable for self-renewal but required 

for proper differentiation of mouse embryonic stem 

(ES) cells 

HaeWon Chung, Bum-Kyu Lee, Nadima Uprety, Wenwen Shen, Jiwoon Lee & Jonghwan Kim * 

Abstract 

Yap1 is a transcriptional co-activator of the Hippo pathway. The 

importance of Yap1 in early cell fate decision during embryogenesis 

has been well established, though its role in embryonic stem (ES) 

cells remains elusive. Here, we report that Yap1 plays crucial roles in 

normal differentiation rather than self-renewal of ES cells. Yap1- 

depleted ES cells maintain undifferentiated state with a typical 

colony morphology as well as robust alkaline phosphatase activity. 

These cells also retain comparable levels of the core pluripotent 

factors, such as Pou5f1 and Sox2, to the levels in wild-type ES cells 

without significant alteration of lineage-specific marker genes. 

Conversely, overexpression of Yap1 in ES cells promotes nuclear 

translocation of Yap1, resulting in disruption of self-renewal and 

triggering differentiation by up-regulating lineage-specific genes. 

Moreover, Yap1-deficient ES cells show impaired induction of 

lineage markers during differentiation. Collectively, our data demonstrate 

that Yap1 is a required factor for proper differentiation of 

mouse ES cells, while remaining dispensable for self-renewal. 

Keywords embryonic stem cells; Yap1; differentiation; Hippo pathway; 

self-renewal 

Subject Categories Signal Transduction; Stem Cells 

DOI 10.15252/embr.201540933 | Received 24 June 2015 | Revised 8 January 

2016 | Accepted 22 January 2016 | Published online 25 February 2016 

EMBO Reports (2016) 17: 519–529 

Introduction 

The Hippo signaling pathway, modulated by cell density and cell– 

cell contact, is implicated in diverse cellular processes including cell 

proliferation [1–5], apoptosis [5,6], and organ size control [7,8]. 

Yap1 is a transcriptional co-activator of the Hippo pathway and is 

known to play a crucial role in the segregation of inner cell mass 

(ICM) and trophectoderm (TE) during early embryogenesis [9–13]. 

While Yap1 resides in the nucleus of trophectodermal cells and 

functions as a critical co-activator for TE development, it is sequestered 

mainly in the cytoplasm of ICM as a phosphorylated inactive 

form due to active Hippo signaling [9]. However, the role of Yap1 in 

ICM is still elusive [9,12]. In addition to Yap1, Taz and Tead family 

members are also crucial players in the Hippo pathway. A 

homologue of Yap1, Taz, shares redundant functions such as 

controlling cell proliferation and sensing mechanical stress [14,15]. 

Furthermore, Tead proteins—important in TE differentiation during 

early embryogenesis, form a complex with Yap1 and are known to 

activate their downstream target genes [16,17]. 

Two different observations on the roles of Yap1 in embryonic 

stem (ES) cells are of note. Recent studies suggested that Yap1 

plays an important role in the maintenance of mouse ES cells as 

an active factor in the nucleus [18,19]. These works showed 

knockdown (KD) of Yap1 promotes differentiation of ES cells while 

overexpression (OE) of Yap1 not only enhances self-renewal but 

also inhibits differentiation of ES cells, even under neuronal differentiation 

conditions [18]. However, the study showing nuclear 

localization of Yap1 in ES cells is somewhat contradictory to the 

function of the Hippo signaling, since mouse ES cells grow as 

tightly packed colonies. It has been suggested that high cell density 

or cell–cell contact activates the Hippo signaling and subsequent 

sequestration of Yap1 in the cytoplasm of various cell lines such 

as HaCaT and NIH-3T3 [1,3,7,20]. Accordingly, another recent 

study has claimed that both Yap1 and Taz are dispensable for 

self-renewal of ES cells in 2i (Gsk3b and Mek inhibitors) culture 

condition [21]. In this case, Yap1- and Taz-depleted ES cells maintained 

undifferentiated state under differentiation-promoting 

culture conditions [21]. Consistent with this observation, studies 

of neuronal differentiation from ES cells have shown that high cell 

density, which activates the Hippo signaling and sequesters Yap1 

in the cytoplasm, blocks differentiation of ES cells [22,23], suggesting 

that nuclear localization of Yap1 might be important in normal 

differentiation of ES cells. 

In the current study, we show that Yap1 is dispensable for the 

maintenance of ES cells but critical in their differentiation. Additional 

testing of Yap1-associated factors including Tead family 

proteins and Taz also supports the dispensability of Yap1 for the 

self-renewal of ES cells. In line with gradual up-regulation of Yap1 

level upon differentiation of ES cells, OE of Yap1 in ES cells 

enhances nuclear abundance of Yap1 accompanied by induction of 

various lineage-specific marker genes. On the contrary, Yap1- 

depleted ES cells showed impaired differentiation. Taken together, 

Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, USA 

*Corresponding author. Tel: +1 512 232 8046; E-mail: jonghwankim@mail.utexas.edu 

ª 2016 The Authors EMBO reports Vol 17 | No 4 | 2016 519

EMBO reports Yap1 is required for differentiation of ES cells HaeWon Chung et al 

our data demonstrate a critical role of Yap1 in normal differentiation 

rather than self-renewal of ES cells. 

Results and Discussion 

Yap1 is dispensable for self-renewal of mouse ES cells 

Previous studies reported that Yap1 is required in the maintenance 

of mouse ES cells by showing that KD of Yap1 promotes 

differentiation of ES cells [18,19]. Conversely, another study 

claimed that double KD of Yap1 and Taz in 2i media does not 

disrupt self-renewal of ES cells [21]. To decipher the roles of Yap1 

in self-renewal and pluripotency of ES cells, we first performed KD 

of Yap1 using lentivirus-delivered shRNAs in J1 mouse ES cells 

(Fig EV1A and Dataset EV1). In contrast to the previous reports 

[18,19], we found that even with > 85% of Yap1 KD, ES cells maintain 

normal colony morphology with high alkaline phosphatase 

(AP) activity, whereas ES cells with KD of Pou5f1 undergo differentiation 

accompanied by loss of AP activity, as expected (Fig 1A). 

We additionally found that these Yap1-depleted ES cells show 

comparable proliferation rate to that of control ES cells (Fig EV1B). 

In agreement with these observations, overall expression levels of 

ES cell core pluripotency factors, such as Pou5f1 and Nanog, as well 

as several lineage-specific regulators were not significantly altered 

upon KD of Yap1 (Figs 1B–D and EV1C). We validated our 

observation by testing two additional ES cell lines (E14 and CJ7) 

and confirmed that KD of Yap1 does not significantly affect features 

of normal self-renewing ES cells (Fig EV2A–F). 

To rule out the possibility of off-target effects and incomplete 

depletion due to shRNA-mediated KD strategies, we additionally 

established Yap1 knockout (KO) ES cell lines harboring premature 

stop codons on both alleles by CRISPR-Cas9-based genome editing 

strategies (Dataset EV1) [24,25]. Consistent with the KD results, 

Yap1 KO ES cells sustained self-renewing status and showed normal 

ES colony morphology and high AP activity, and levels of 

pluripotency-related genes were comparable to those of wild-type 

ES cells (Figs 1E–G and EV2G–L). Yap1 KO ES cells were able 

to maintain self-renewal for more than 1 month in culture 

(Fig EV3A–C). Taken together, these results indicate that Yap1 is 

dispensable for self-renewal of mouse ES cells. 

To further validate the dispensability of Yap1 in self-renewal of 

ES cells, we sought to monitor the global gene expression profiles of 

Yap1 KD and Yap1 KO ES cells using RNA-seq approaches. As 

expected, comparison of expression profiles between ES and differentiating 

ES cells revealed many differentially expressed genes 

(DEGs) (Fig 1H and Dataset EV2). However, expression levels of 

these genes were not altered significantly upon KD or KO of Yap1 

(Fig 1H) which was further confirmed by RT–qPCR (Fig 1I). Overall, 

these results indicate that the depletion of Yap1 does not trigger 

differentiation of ES cells. 

Unlike differentiating ES cells, a hierarchical clustering of 

global expression data revealed that Yap1 KD and Yap1 KO ES 

cells were clustered together with normal and control ES cells, 

indicating that Yap1-deficient ES cells have similar expression profiles 

to those of normal ES cells (Fig 1J). We also investigated the 

activity of previously defined functional modules in ES cells (Core 

and PRC) [26]. Module activity is defined as an averaged 

expression of all genes in each module. Briefly, the Core module 

includes core pluripotency factors such as Pou5f1, Nanog, and 

Sox2, most of which are highly expressed in self-renewing 

ES cells. On the other hand, the PRC module includes many 

lineage-specific regulators, such as Fgf5, Bmp4, and Hand1, most 

of which are repressed in ES cells. Since differentiation of ES cells 

decreases the activity of Core module but increases the activity of 

PRC module [26], we sought to examine module activities upon 

KD or KO of Yap1 to test whether cells maintain self-renewal. As 

shown in Fig 1K, ES cells with depleted Yap1 did not show 

down-regulation of Core module activity or up-regulation of PRC 

module activity, suggesting that Yap1-depleted ES cells largely 

maintain self-renewing and undifferentiated states. Further correlation 

analyses verified that the global expression patterns of 

Yap1-depleted ES cells showed higher correlation with those of 

control ES cells (R 2 = 0.978 for Yap1 KD, R 2 = 0.977 for Yap1 

KO1, and R 2 = 0.979 for Yap1 KO2) than differentiating ES cells 

(R 2 = 0.781) (Fig 1L). Collectively, these data provide strong 

evidence that the depletion of Yap1 does not significantly alter 

the self-renewal of ES cells. 

Figure 1. Yap1 is dispensable for self-renewal of J1 mouse ES cells (see also Figs EV1–EV3). 

A Colony morphology and alkaline phosphatase (AP) activity of ES cells upon KD of Yap1 and Pou5f1. KD1 and KD2 indicate two different shRNA sequences tested. All 

the following cell morphology and AP staining pictures were taken two passages (4 days) after lentivirus infection unless otherwise stated. 

B, C mRNA expression levels of Pou5f1, Nanog, Sox2, Esrrb (B), and Yap1 (C) upon KD of Yap1. All the following mRNA samples were harvested 4 days after lentivirus 

infection while passaged every 2 days unless otherwise stated. Data are represented as mean SD. 

D Protein levels of Yap1, Pou5f1, and Nanog upon KD of Yap1. All the following protein samples were harvested 4 days after lentivirus infection while passaged every 

2 days unless otherwise stated. 

E Colony morphology and AP activity of mouse embryonic stem cells (ESC) and three Yap1 KO clones (KO1-KO3). 

F mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb upon KO of Yap1. Data are represented as mean SD. 

G Protein levels of Yap1, Pou5f1, and Nanog in Yap1 KO clones. 

H A heatmap showing relative mRNA expression levels of 3,605 genes differentially expressed (> twofold) between ES cells and differentiating ES cells (dESC). Genes 

were sorted by the fold changes of gene expression between dESC and ES cells. Corresponding gene expression profiles obtained from Yap1 KO1, Yap1 KO2, and 

Yap1 KD cells are also shown. 

I mRNA expression levels of lineage-specific marker genes upon KD of Yap1. dESC were used as control cells. 

J A heatmap showing Pearson’s correlation coefficients of gene expression profiles obtained from ESC, control virus-infected ES cells (Control), dESC, Yap1 KD cells, 

and Yap1 KO cells. 

K Relative average module activities (Core and PRC) in Yap1 KD1 cells, KO cells, and dESC. Module activities were normalized by the data obtained in ES cells. Data 

are represented as mean SEM. 

L Scatter plots showing log 10 (FPKM) values of genes in Yap1 KD1 cells and Control (upper left panel), dESC and ESC (bottom left panel), and Yap1 KO cells and ES 

cells (right two panels). Pearson’s correlation coefficients (R 2 ) are indicated. “FPKM” indicates fragments per kilobase of transcript per million fragments mapped. 

▸ 

520 

EMBO reports Vol 17 | No 4 | 2016 ª 2016 The Authors

HaeWon Chung et al Yap1 is required for differentiation of ES cells EMBO reports 

A Control Yap1 KD1 Yap1 KD2 Pou5f1KD 

AP staining Bright field 

AP staining Bright field 

200µm 200µm 200µm 

200µm 

200µm 200µm 200µm 

200µm 

E 

ESC Yap1 KO1 Yap1 KO2 Yap1 KO3 

200µm 200µm 200µm 200µm 

200µm 200µm 200µm 200µm 

Relative gene expression 

2 

1 

0 

B 

F 


Pou5f1 

Nanog 

Sox2 

Esrrb 

Control 

1.5 

1 

0.5 

Yap1 KD1 

Yap1 KD2 

ESC 

Yap1 KO1 

dESC 

C 

Relative Yap1 expression 

Yap1 KO2 

Yap1 KO3 

0 

Pou5f1Nanog Sox2 Esrrb 

1 

0 

Control 

Yap1 KD1 

Yap1 KD2 

D 

Yap1 

Pou5f1 

G 

Yap1 

Pou5f1 

Nanog 

Gapdh 

Nanog 

Gapdh 

Control 

ESC 

Yap1 KO1 

Yap1 KD1 

Yap1 KD2 

Yap1 KO2 

Yap1 KO3 

H 

J 

1,826 genes 

1,779 genes 

ESC 

Yap1 KO1 

Yap1 KO2 

Control 

Yap1 KD1 

dESC 

dESC 

Yap1 KO1 

Yap1 KO2 

Yap1 KD1 

ESC 

Yap1 KO1 

Yap1 KO2 

Control 

Yap1 KD 1 

dESC 

log2 

+3.0 

- 3.0 

1.0 

0.8 

0.6 

0.4 

I 


K 

Module activity 

1000 

100 

10 

1 

0 

1.4 

0.7 

0 

-0.7 

Yap1 KO1 

Control 

Yap1 KD1 

Yap1 KD2 

dESC 

Gbx2 Nes Gata2 T Sox17 Nodal Cdx2 Gata3 

Core 

PRC 

Yap1 KO2 

Yap1 KD1 

dESC 

L 

Yap1 KD1 

dESC 

-2 

-2 

4 

2 

0 

-2 

4 

2 

0 

-2 

0 

0 

R² = 0.978 

Control 

2 4 

R² = 0.781 

2 4 

ESC 

Yap1 KO1 

Yap1 KO2 

-2 

-2 

4 

2 

0 

0 

-2 

4 

2 

-2 

0 

0 

R² = 0.977 

2 4 

ESC 

R² = 0.979 

2 4 

ESC 

Figure 1. 

ª 2016 The Authors EMBO reports Vol 17 | No 4 | 2016 

521


Taz and Tead family proteins are not required for self-renewal of 

ES cells and do not compensate Yap1 functions in Yap1-depleted 

ES cells 

Taz is homologous to Yap1 and has similar functions to Yap1, such 

as regulation of proliferation and activation of TE lineage markers 

[14,27]. To rule out the possibility of compensation by Taz in Yap1 

KD ES cells, we performed both single and double KD of Yap1 and 

Taz using shRNAs under drug selections (blasticidin and puromycin, 

respectively). J1 ES cells with depletion of both Yap1 and 

Taz (> 85% of KD for each) maintained typical colony morphology 

as well as high AP activity (Fig 2A and B). Additionally, the levels 

of pluripotency markers, such as Pou5f1 and Nanog, as well as various 

lineage markers were not significantly affected by either single 

or double KD of Yap1 and Taz (Fig 2C and D), indicating that the 

dispensability of Yap1 in self-renewing ES cells is not due to the 

compensatory effect of Taz. 

Since Yap1 is known to require Tead family proteins to activate 

its downstream target genes in NIH-3T3 and MCF10A cell lines 

[17,28,29], we investigated whether Tead proteins are also 

dispensable for the maintenance of ES cells. To do so, we first 

performed KD of Tead2 in ES cells. In contrast to the previous 

report [19], we did not observe any significant alteration of cell 

morphology or reduced AP activity upon down-regulation of Tead2 

(at least > 90% of KD in mRNA levels) (Fig EV4A and B). In 

accordance with the colony morphology, Tead2 KD ES cells 

expressed similar levels of pluripotency genes compared to wildtype 

ES cells (Fig EV4C and D). These results were confirmed by 

generation of three independent Tead2 KO ES cell clones using 

CRISPR-Cas9 strategies (Dataset EV1). These Tead2 KO clones also 

maintained self-renewal without differentiation (Fig EV4E–G). We 

additionally conducted triple KD of Tead1/3/4 with triple drug 

selection (at least > 80% of KD for each), and did not observe any 

significant alteration of cell morphology or AP activity which is in 

contrast to the previous report [18] (Fig 2E and F). Similar to the 

results obtained from the KD of Yap1, triple Tead KD ES cells 

expressed comparable levels of pluripotency genes shown in wildtype 

ES cells without any significant activation of lineage-specific 

regulators (Fig 2G–I). The results were further validated by double 

KD of Tead1/3 in Tead4 KO cells. ES cells with Tead4 KO and 

Tead1/3 KD also maintained self-renewal (Fig EV4H–J). Collectively, 

our data suggest that both Yap1 and Yap1-associated 

proteins such as Taz and Tead are not required for self-renewal of 

ES cells. 

Yap1 is induced and translocated into the nucleus upon 

differentiation of ES cells 

In order to investigate the roles of Yap1 in differentiation of ES cells, 

we examined the expression level of Yap1 in self-renewing mouse 

ES cells as well as upon differentiation of ES cells. Analysis of 

published mRNA expression data obtained upon time-course differentiation 

of embryoid body (EB) [30] revealed that Yap1 is 

moderately expressed in ES cells while its expression gradually 

increases upon differentiation (Fig 3A). We differentiated mouse J1 

ES cells by the withdrawal of leukemia inhibitory factor (LIF) in the 

culture media and examined the level of Yap1. Consistent with the 

results from the EB differentiation, both mRNA and protein levels of 

Yap1 were moderately increased upon spontaneous differentiation 

(Fig 3B and C). 

Since active Hippo signaling leads to phosphorylation and 

cytoplasmic sequestration of Yap1, blocking Yap1’s function as a 

transcriptional coactivator [7,9,12], we examined the levels of phospho-Yap1 

and its subsequent localization in both self-renewing and 

differentiating ES cells. Western blot analysis showed that Yap1 is 

highly phosphorylated in self-renewing ES cells, but the level of 

phospho-Yap1 is reduced in differentiating ES cells (Fig 3D). Given 

the fact that phospho-Yap1 is sequestered in the cytoplasm [7,9,12], 

we examined Yap1 localization by immunofluorescence (IF). 

Consistent with hyper-phosphorylation of Yap1 in ES cells, IF results 

revealed that Yap1 resides primarily in the cytoplasm of selfrenewing 

ES cells (Fig 3E–G). However, upon differentiation of 

multiple mouse ES cell lines we tested (J1, CJ7, and E14), Yap1 was 

translocated into the nucleus (Figs 3E–G and EV5A–D). Cytoplasmic 

Yap1 in ES cells could be attributed to compact ES cell colonies with 

active Hippo signaling [7], while lower cell density of differentiating 

ES cells growing in a monolayer leads to inactive Hippo signaling, 

resulting in the nuclear localization of Yap1. 

We further investigated the activity of nuclear Yap1 using a 

synthetic Yap1-responsive luciferase (8xGTIIC) construct as previously 

designed for the measurement of Yap1 transcriptional activity 

in mechanical stress condition (Fig 3H) [15,19,31,32]. The luciferase 

construct contains repeated Yap1-Tead binding motifs (eight times) 

in front of the minimal cTNT promoter followed by a luciferase 

reporter gene [15,32,33]. As shown in Fig 3I and J, we observed a 

significant increase in luciferase activity in both ES cells with transient 

OE of Yap1 and in differentiating ES cells compared to the 

reporter activity in self-renewing ES cells, thereby indicating the 

increased level of nuclear Yap1 either by OE of Yap1 or by ES cell 

differentiation promotes transcription of the reporter gene. An 

induced level and nuclear localization of Yap1 were also confirmed, 

along with the increased Yap1 activity in Pou5f1 KD ES cells 

undergoing TE differentiation (Fig 3K–M). 

Yap1 is required for normal differentiation of ES cells 

As we observed increased expression levels and nuclear localization 

of Yap1 in differentiating ES cells (Fig 3), we hypothesized that 

Yap1 may have critical roles in differentiation of ES cells. To address 

this, we tested differentiation potential of Yap1 KD ES cells. Upon 

3 days of differentiation, completely differentiated and monolayered 

cellular morphology with reduced AP activity were observed 

in control ES cells. However, Yap1 KD cells maintained typical 

colony morphology with high AP activity comparable to that of selfrenewing 

ES cells even after 2–3 days of differentiation (Fig 4A). 

We further found that expression levels of some pluripotency factors 

such as Sox2 and Esrrb were relatively highly maintained in Yap1- 

depleted cells upon differentiation, although the expression of other 

core factors, Pou5f1 and Nanog, was decreased similarly to their 

levels in control cells upon differentiation (Fig EV6A). Moreover, 

up-regulation of various lineage-specific markers, such as Nes, T, 

Gsc, Gata6, Cdx2, and Gata3, was significantly impaired during 

differentiation of Yap1-depleted cells (Fig EV6B), suggesting that the 

depletion of Yap1 affects differentiation potential of ES cells. 

In order to get further insight into the roles of Yap1 in global 

transcriptional regulation during differentiation, we analyzed gene 

522 



A B C 

Control Taz KD Yap1 KD Taz/Yap1 KD 

Taz Yap1 

1.5 

D 


Bright field 

AP staining 

2 

1.5 

1 

0.5 

0 

Control 

Taz KD 

200µm 200µm 200µm 200µm 

200µm 200µm 200µm 200µm 

Yap1 KD 

Taz/Yap1 KD 


1 

0.5 

0 

Control 

Taz KD 

Yap1 KD1 

Taz/Yap1 KD 

Gbx2 Nes Gata2 T Nodal Gata3 Gsc Amot Mixl1 Fzd6 Edn1 Cited1 Msx2 Krt18 


2 

1.5 

1 

0.5 

Control 

Taz KD 

Yap1 KD 

Taz/Yap1 KD 

0 

Pou5f1 Nanog Sox2 Esrrb 

E F G 

Control Tead1/3/4 KD 

Tead1 

Tead3 

Tead4 

1 

I 

Bright field 

AP staining 

2 

Control 

200µm 200µm 

200µm 200µm 

Tead 1/3/4 KD 

Relative gene expresison 

0.5 

0 

Control 

Tead1/3/4 KD 


1 

0.5 

0 

Control 

Tead1/3/4 KD 

Pou5f1 

Nanog 

Sox2 

Esrrb 

H 

Tead1 

Tead3 

Tead4 

Pou5f1 

Gapdh 

Control 

Tead1/3/4 KD 

N.S. 


1.5 

1 

0.5 

0 

Gbx2 

Gata2 

T 

Sox17 

Nodal 

Cdx2 

Gata3 

Isl1 

Gsc 

Gata4 

Gata6 

Pitx2 

Eomes 

Sox13 

Tgfb2 

Amot 

Mixl1 

Fzd6 

Edn1 

Cited1 

Msx2 

Krt18 

Figure 2. Taz and Tead family proteins are not required for the self-renewal of J1 mouse ES cells (see also Fig EV4). 

A Colony morphology and AP activity of ES cells upon KD of Yap1 and Taz. 

B–D mRNA expression levels of Yap1 and Taz (B), Pou5f1, Nanog, Sox2, and Esrrb (C), and lineage-specific marker genes (D) upon KD of Yap1 and Taz. Data are 

represented as mean SD. 

E Colony morphology and AP activity of ES cells upon KD of Tead1/Tead3/Tead4. 

F, G mRNA expression levels of Tead1, Tead3, and Tead4 (F) and Pou5f1, Nanog, Sox2, and Esrrb (G) upon KD of Tead 1/3/4. Data are represented as mean SD. 

H Protein levels of Tead1, Tead3, Tead4, and Pou5f1 upon KD of Tead1/Tead3/Tead4. N.S., non-specific. 

I mRNA expression levels of lineage-specific marker genes upon KD of Tead1/Tead3/Tead4. Data are represented as mean SD. 


523


A 


2 Yap1 

Pou5f1 

1.5 Gata6 

1 

0.5 

0 

0 

0.25 

0.5 

0.75 

1 

1.5 

2 

4 

7 

B 


2 

1 

0 

ESC 

dESC 

C 

Yap1 

Pou5f1 

Actb 

D 

p-Yap1 

Yap1 

Time (days) 

0 2 4 6 

0 2 4 

Time (Days) 

E 

ESC 

dESC 

F 

ESC 

dESC 

G 

ESC 

dESC 

Yap1 

20um 

20um 

20um 

20um 

20um 

20um 

Merge DAPI 

20um 

20um 

Merge DAPI 

20um 

20um 

Merge DAPI 

20um 

20um 

20um 

20um 

20um 

20um 

20um 

20um 

Merge 

Merge 

Merge 

Yap1 

Yap1 

5um 5um 5um 5um 5um 5um 

H 

Yap1 

Tead 

8xGTIIC promoter luciferase 

Relative luc activity 

I 

12 

9 

6 

3 

0 

Control 

** 

Yap1 OE 

J 


5 

4 

3 

2 

1 

0 

ESC 

K 

** ** 

6 

dESC 


4 

2 

0 

Control 

Pou5f1 KD 

L 

Control 

Pou5f1 KD 

Yap1 DAPI Merge 

20um 20um 20um 

20um 20um 20um 

M 


3 

2 

1 

0 

Control 

** 

Pou5f1 KD 

Figure 3. 

expression profiles obtained from RNA-seq of normal ES cells 

and Yap1-depleted ES cells before and after differentiation. As 

shown in Fig 4B, gene expression patterns of DEGs (Yap1 KD ES 

cells/wild-type ES cells) upon differentiation showed an inverse 

correlation with the expression patterns of wild-type differentiating 

cells over self-renewing ES cells (Dataset EV3). The heatmap results 

524 



◀ 

Figure 3. Yap1 is up-regulated and translocated into nucleus during ES cell differentiation (see also Fig EV5). 

A Relative mRNA levels of Yap1, Pou5f1, and Gata6 during time-course embryoid body (EB) differentiation. Gene expression data were obtained from GSE3749. Pou5f1 

and Gata6 serve as representative ES cell marker and lineage-specific marker, respectively. 

B Relative Yap1 mRNA levels in ES cells (ESC) and differentiating ES cells (dESC) (LIF withdrawal for 4 days) and data are represented as mean SD. To differentiate 

ES cells, cells were incubated in LIF-withdrawn medium for 4 days. Both ESC and dESC were passaged every 2 days. 

C Protein levels of Yap1 and Pou5f1 during time-course differentiation upon LIF withdrawal. 

D Phospho-Yap1 levels during time-course differentiation. Samples were normalized by total Yap1 level. 

E–G Immunofluorescence (IF) images depicting localization of Yap1 in J1 (E), CJ7 (F), and E14 (G) mouse ESC (top) and dESC (bottom). The white arrow indicates 

nucleolus. Bottom panels represent higher magnification of the above panels. Dashed circle indicates nucleus border. 

H A schematic diagram depicting a Yap1-responsive luciferase reporter (8xGTIIC) construct. 

I Luciferase reporter assay using Yap1-responsive luciferase reporter (8xGTIIC) upon transient overexpression (OE) Yap1 in ES cells. P-values were calculated using 

Student’s t-test. Data are represented as mean SD. **P < 0.01. “Control” indicates ES cells infected with control virus not expressing any specific shRNA 

sequence. 

J Relative activity of Yap1-responsive luciferase reporter gene in ESC and dESC. P-values were calculated using Student’s t-test. Data are represented as mean SD. 

**P < 0.01. 

K Relative Yap1 mRNA levels in Control and Pou5f1 KD ES cells. Data are represented as mean SD. **P < 0.01. “Control” indicates ES cells infected with control 

virus not expressing any specific shRNA sequence. 

L IF images depicting localization of Yap1 in Control and Pou5f1 KD ES cells. 

M Relative activity of Yap1-responsive luciferase reporter gene upon Pou5f1 KD in ES cells. P-values were calculated using Student’s t-test. Data are represented as 

mean SD. **P < 0.01. 

clearly revealed that Yap1-depleted ES cells are not properly differentiated. 

Additional analyses of the Core and PRC module activity 

consistently indicated that Yap1 depletion causes stronger Core 

module activity with weaker PRC module activity during differentiation, 

indicating that KD of Yap1 delayed or impaired proper differentiation 

of ES cells (Fig 4C). Gene ontology (GO) term analysis using 

the genes that are not properly induced in Yap1-depleted cells 

compared to wild-type ES cells upon differentiation also revealed 

that these genes are implicated in various development-related 

processes, such as blood vessel development, chordate embryonic 

development, and in utero embryonic development (Fig EV6C). 

These collectively demonstrate that adequate levels of Yap1 are 

critical in normal differentiation of ES cells. 

Ectopic expression of Yap1 in ES cells is sufficient to induce 

up-regulation of lineage marker genes 

To further test roles of Yap1 in ES cell differentiation, we performed 

OE of Yap1 in ES cells. Yap1 mainly resides in the cytoplasm of selfrenewing 

ES cells. While OE of Yap1 increases both nuclear and 

cytoplasmic Yap1 levels, we detected more nuclear Yap1 in Yap1 OE 

cells, indicating that exogenous Yap1 can translocate into the 

nucleus and act on its target genes (Fig EV6D and E). Yap1 OE cells 

also showed flattened morphology similar to that of differentiating 

ES cells with reduced AP activity even in the presence of LIF 

(Fig 4D). We further examined global gene expression profiles of 

Yap1 OE cells, and a clustering analysis showed that the DEGs upon 

OE of Yap1 (Yap1 OE/ES cells) are highly similar to the DEGs of 

differentiating ES cells over self-renewing ES cells (Fig 4E and 

Dataset EV4). Consistently, the activity of the Core module was 

significantly decreased upon OE of Yap1, while the PRC module 

activity was dramatically increased (Figs 4F and EV6F). These 

results suggest that OE of Yap1 is sufficient to trigger ES cell 

differentiation. Additional GO term analysis revealed that genes 

up-regulated upon OE of Yap1 are significantly enriched in developmental 

processes, such as chordate embryonic development, 

skeletal system development, and embryonic organ development 

(Fig 4G), further demonstrating that the ectopic expression of Yap1 

promotes differentiation of ES cells. 

Unlike the well-established functions of the Hippo signaling 

pathway in the first cell fate decision, the roles of Yap1 in ES cells, 

ICM, and during differentiation of ES cells or ICM are still not well 

understood. Here, we reveal that Yap1, a transcriptional effector of 

Hippo pathway, is a crucial factor implicated in differentiation 

rather than self-renewal of ES cells. In contrast to the previous 

reports [18,19], a depletion of Yap1 does not show any significant 

effect on the maintenance of multiple ES cells we tested. This is 

consistent with the nonessential roles of Yap1 in ES cells grown 

Figure 4. Yap1 is required for differentiation of ES cells (see also Fig EV6). 

A Colony morphology and AP activity of Control and Yap1 KD ES cells upon differentiation. Morphology and AP staining pictures were taken 2 days after differentiation. 

B A heatmap showing relative mRNA expression levels of 1,995 genes differentially expressed (> twofold) between Yap1 KD ES cells and Control upon 4 days of 

differentiation. Genes were sorted by the fold changes of gene expression between Yap1 KD ES cells and Control (first column). Corresponding gene expression 

changes between ES cells (ESC) and differentiating ES cells (dESC) are shown in the second column. 

C Relative average module activities (Core and PRC modules) between Yap1 KD ES cells and Control cells upon differentiation. Data are represented as mean SEM. 

D Colony morphology and AP activity in Yap1 OE cells. Two different Yap1 OE clones (OE1 and OE2) and pool of Yap1 OE (OE pool) were used. Cell morphology and AP 

staining pictures were taken 3 weeks after electroporation. 

E A heatmap showing relative mRNA expression levels of 2,137 genes differentially expressed (> twofold) between Yap1 OE ES cells and control ES cells. Genes were 

sorted by the fold changes of gene expression between Yap1 OE ES cells and control ES cells (first column) and corresponding gene expression profiles obtained from 

dESC are shown. 

F Relative average module activities (Core and PRC modules) between Yap1 OE cells and control cells are shown. Data are represented as mean SEM. 

G Genes up-regulated in Yap1 OE cells were tested using David 6.7. Significantly enriched gene ontology (GO) terms (biological functions) are shown. Developmental 

process-related GO terms are highlighted in red. 

▸ 


525

= 


A B C 

Bright field AP staining 

Yap1 KD1/Control 

(Differentiation) 

dESC/ESC 

Control 

Yap1 KD 

Control 

Yap1 OE1 

Yap1 OE2 

Yap1 OE 

pool 

200µm 

200µm 

D 

Bright field 

200µm 

200µm 

200µm 

200µm 

200µm 

200µm 

AP staining 

200µm 

200µm 

200µm 

200µm 

1,269 genes 868 genes 

1,450 genes 545 genes 

E 

Yap1 OE1 

dESC 

log2 

+3.0 

-3.0 -0.4 

log2 

+3.0 

-3.0 



0.4 

0.2 

0 

-0.2 

F 

0.4 

0.2 

0 

-0.2 

Core 

Core 

PRC 

PRC 

G 

chordate embryonic development 

skeletal system development 

regulation of cell proliferation 

embryonic organ development 

positive regulation of 

developmental process 

embryonic morphogenesis 

heart development 

vasculature development 

in utero embryonic development 

tube development 

Biological process 

0 2 4 6 8 

-log10(P-value) 

Figure 4. 

526 



under the 2i condition [21]. In agreement with the dispensability 

of Yap1 in ES cells, other key effectors of the Hippo pathway 

(Tead family proteins and Taz) do not compensate Yap1, and are 

also not necessary for the maintenance of ES cells, implying that 

the transcriptional effectors of the Hippo pathway are at least 

dispensable for self-renewal of mouse ES cells. In addition, we 

show that Yap1 is mainly sequestered in the cytoplasm of selfrenewing 

ES cells, while it localizes in the nucleus upon 

differentiation. The predominant nuclear localization of Yap1 in 

differentiating ES cells may be due to inactive Hippo signaling in 

the cells growing with lower density. Consistently, OE of Yap1 in 

ES cells triggers nuclear localization of Yap1 and induces differentiation 

along with activation of diverse lineage markers. Our global 

expression analyses further suggest that Yap1 may promote 

differentiation by activating differentiation-related genes rather 

than repressing pluripotency-related genes, which is consistent 

with the observation of Yap1 KO embryo, which dies around E10 

due to the defects in yolk sac vasculogenesis, chorioallantoic 

fusion, and embryonic axis elongation [34]. Notably, a recent 

study done in human ES cells suggested that YAP1 represses 

mesendoderm differentiation [35], possibly due to the differences 

in signaling pathways between human and mouse ES cells [36,37]. 

In-depth investigation of the impaired regulation of three lineagespecific 

genes as well as TE-related genes in Yap1 KD ES cells 

upon differentiation may provide further insights into the functions 

of Yap1 in early embryogenesis. Together, our data establish that 

Yap1 is a critical regulator for proper differentiation but 

dispensable for self-renewal of ES cells. 

Materials and Methods 

Cell culture 

J1, E14, and CJ7 mouse ES cells were maintained in Dulbecco’s 

modified Eagle’s medium (DMEM, Gibco Ref. 11965) supplemented 

with 18% of fetal bovine serum (FBS), penicillin/streptomycin/ 

L-glutamine (Gibco Ref. 10378), MEM nonessential amino acid 

(Gibco Ref. 11140), nucleosides (Millipore Cat. ES-008-D), 100 lM 

b-mercaptoethanol (Sigma M3148), and 1,000 U/ml recombinant 

leukemia inhibitory factor (LIF, Millipore Cat. ESG1107). ES cells 

were cultured on 0.1% gelatin-coated plates at 37°C and 5% CO 2 

and passaged every 2 days. HEK 293T cells were maintained in 

DMEM supplemented with 10% of FBS and penicillin/streptomycin/ 

L-glutamine. To differentiate ES cells, cells were washed three times 

with the media without LIF and then incubated for 4 days while 

passaging every 2 days. 

shRNA lentiviral production and infection 

12-well plate with virus containing media supplemented with 

10 lg/ml polybrene (Millipore Cat. TR-1003-G). After 1 day of infection, 

cells are selected with appropriate antibiotics and passaged 

every 2 days. Cell morphology, AP staining, protein and mRNA 

levels were examined two passages after the infection. 

Luciferase reporter gene assay 

For the luciferase reporter gene assay, 2.5 × 10 5 J1 ES cells in each 

24 well were co-transfected with 100 ng of the GTIIC vector, 5 ng of 

PGL4.75 vector containing a Renilla reporter gene as an internal 

control reporter using Lipofectamine 3000 (Life Technologies, Cat. 

L3000008) and then cultivated for 24 h. To measure luciferase 

reporter gene activity, cells were washed two times with PBS, lysed, 

and the luciferase activities were measured using the Dual 

Luciferase assay kit (Promega, E1910). 

Immunofluorescence 

~3 × 10 5 /ml ES cells were plated on 0.1% gelatin pre-coated 

l-Slide VI 0.4 (Ibidi Cat. 80606). Slides were fixed with 3.7% 

paraformaldehyde for 15 min at room temperature and permeabilized 

with 0.5% Triton X-100 for 10 min. Slides were then incubated 

with blocking solution (3% BSA and 1% normal horse serum in 

PBS) for 1 h at room temperature, Yap1 primary antibody solution 

(1:200 dilution, Santa Cruz sc-101199) overnight at 4°C, and 

secondary antibody solution (1:1,000 dilution) conjugated to Alexa 

Fluor 488 for 1 h at room temperature. Lastly, slides were mounted 

with ProLong Gold antifade reagent with DAPI (Molecular Probes 

P36935) and imaged on a Zeiss 710 laser scanning confocal and 

structured illumination microscope. 

RNA sequencing and data processing 

One lg of RNAs was used to generate Illumina-compatible sequencing 

libraries using mRNA isolation kit (NEB, E7490L) and RNA 

library prep kit (NEB, E7530S) according to the manufacturer’s 

protocol. Adapter ligation was done with sample-specific barcodes. 

RNA-seq libraries were sequenced using an Illumina NextSeq 500 

machine. Single-end reads from RNA-seq were mapped onto the 

mouse genome assembly (mm9) using default setting of Tophat2. 

Transcript-level expression analysis was performed using Cuffdiff to 

calculate FPKM (fragments per kilobase of transcript per million 

mapped reads) [38]. 

Data deposition 

The RNA-seq data used in this study were deposited at the Gene 

Expression Omnibus (GEO) under the accession number GSE69669. 

HEK 293T cells were plated at ~6 × 10 6 cells per 100 mm 2 and then 

transfected with 6 lg of pLKO.1 shRNA vector (Sigma) (Dataset 

EV1), 4 lg of pCMV-D8.9, and 2 lg of VSVG plasmids using 30 ll of 

Fugene 6 (Promega Ref. 2692), according to the manufacturer’s 

protocol. After 24 h, HEK 293T medium was replaced with ES 

medium. Two days after transfection, supernatant containing viral 

particles was collected and filtered through 0.45-lm Supor 

membrane (PALL Ref. 4654). ~2 × 10 5 ES cells were plated on 

Expanded View for this article is available online. 

Acknowledgements 

We thank all the members of Kim laboratory for their help and support and 

the ICMB Microscopy and Imaging Facility as well as the Genome Sequencing 

and Analysis Facility (GSAF) at UT-Austin. The project is supported by 

R01GM112722 from NIH/NIGMS and R1106 from the Cancer Prevention 

Research Institute of Texas (CPRIT) to J.K. J.K. is a CPRIT scholar. 


527


Author contributions 

HWC, NU, and WS performed the experiments. HWC and B-KL analyzed 

RNA-seq data. HWC, B-KL, JL, and JK conceived work and wrote the 

manuscript. 

Conflict of interest 

The authors declare that they have no conflict of interest. 

References 

1. Kim N, Koh E (2011) E-cadherin mediates contact inhibition of proliferation 

through Hippo signaling-pathway components. Proc Natl Acad Sci 

USA 2011: 11930 – 11935 

2. Schlegelmilch K, Mohseni M, Kirak O, Pruszak J, Rodriguez JR, Zhou D, 

Kreger BT, Vasioukhin V, Avruch J, Brummelkamp TR et al (2011) Yap1 

acts downstream of a-catenin to control epidermal proliferation. Cell 

144: 782 – 795 

3. Mori M, Triboulet R, Mohseni M, Schlegelmilch K, Shrestha K, 

Camargo FD, Gregory RI (2014) Hippo signaling regulates microprocessor 

and links cell-density-dependent miRNA biogenesis to cancer. Cell 

156: 893 – 906 

4. Silvis MR, Kreger BT, Lien W-H, Klezovitch O, Rudakova GM, Camargo 

FD, Lantz DM, Seykora JT, Vasioukhin V (2011) a-catenin is a tumor 

suppressor that controls cell accumulation by regulating the localization 

and activity of the transcriptional coactivator Yap1. Sci Signal 4: ra33 

5. Huang J, Wu S, Barrera J, Matthews K, Pan D (2005) The Hippo signaling 

pathway coordinately regulates cell proliferation and apoptosis by inactivating 

Yorkie, the Drosophila homolog of YAP. Cell 122: 421 – 434 

6. Lee K-K, Yonehara S (2012) Identification of mechanism that couples 

multisite phosphorylation of Yes-associated protein (YAP) with transcriptional 

coactivation and regulation of apoptosis. J Biol Chem 287: 

9568 – 9578 

7. Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J, Xie J, Ikenoue T, Yu J, Li L 

et al (2007) Inactivation of YAP oncoprotein by the Hippo pathway is 

involved in cell contact inhibition and tissue growth control. Genes Dev 

21: 2747 – 2761 

8. Camargo FD, Gokhale S, Johnnidis JB, Fu D, Bell GW, Jaenisch R, Brummelkamp 

TR (2007) YAP1 increases organ size and expands undifferentiated 

progenitor cells. Curr Biol 17: 2054 – 2060 

9. Nishioka N, Inoue K, Adachi K, Kiyonari H, Ota M, Ralston A, Yabuta N, 

Hirahara S, Stephenson RO, Ogonuki N et al (2009) The Hippo signaling 

pathway components Lats and Yap pattern Tead4 activity to distinguish 

mouse trophectoderm from inner cell mass. Dev Cell 16: 398 – 410 

10. Hirate Y, Hirahara S, Inoue K-I, Suzuki A, Alarcon VB, Akimoto K, Hirai T, 

Hara T, Adachi M, Chida K et al (2013) Polarity-dependent distribution 

of angiomotin localizes Hippo signaling in preimplantation embryos. 

Curr Biol 23: 1181 – 1194 

11. Rayon T, Menchero S, Nieto A, Xenopoulos P, Crespo M, Cockburn K, 

Cañon S, Sasaki H, Hadjantonakis A-K, de la Pompa JL et al (2014) 

Notch and hippo converge on Cdx2 to specify the trophectoderm 

lineage in the mouse blastocyst. Dev Cell 30: 410 – 422 

12. Cockburn K, Biechele S, Garner J, Rossant J (2013) The Hippo pathway 

member Nf2 is required for inner cell mass specification. Curr Biol 23: 

1195 – 1201 

13. Wicklow E, Blij S, Frum T, Hirate Y, Lang RA, Sasaki H, Ralston A (2014) 

HIPPO pathway members restrict Sox2 to the inner cell mass where it 

promotes ICM fates in the mouse blastocyst. PLoS Genet 10: e1004618 

14. Imajo M, Ebisuya M, Nishida E (2014) Dual role of YAP and TAZ in 

renewal of the intestinal epithelium. Nat Cell Biol 17: 7 – 19 

15. Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato 

F, Le Digabel J, Forcato M, Bicciato S et al (2011) Role of YAP/TAZ 

in mechanotransduction. Nature 474: 179 – 183 

16. Ota M, Sasaki H (2008) Mammalian Tead proteins regulate cell proliferation 

and contact inhibition as transcriptional mediators of Hippo 

signaling. Development 135: 4059 – 4069 

17. Li Z, Zhao B, Wang P, Chen F, Dong Z, Yang H, Guan KL, Xu Y (2010) 

Structural insights into the YAP and TEAD complex. Genes Dev 24: 

235 – 240 

18. Lian I, Kim J, Okazawa H, Zhao J, Zhao B, Yu J, Chinnaiyan A, Israel MA, 

Goldstein LSB, Abujarour R et al (2010) The role of YAP transcription 

coactivator in regulating stem cell self-renewal and differentiation. 

Genes Dev 24: 1106 – 1118 

19. Tamm C, Böwer N, Annerén C (2011) Regulation of mouse embryonic 

stem cell self-renewal by a Yes-YAP-TEAD2 signaling pathway downstream 

of LIF. J Cell Sci 124: 1136 – 1144 

20. Varelas X, Samavarchi-Tehrani P, Narimatsu M, Weiss A, Cockburn K, 

Larsen BG, Rossant J, Wrana JL (2010) The Crumbs complex couples cell 

density sensing to Hippo-dependent control of the TGF-b-SMAD pathway. 

Dev Cell 19: 831 – 844 

21. Azzolin L, Panciera T, Soligo S, Enzo E, Bicciato S, Dupont S, Bresolin S, 

Frasson C, Basso G, Guzzardo V et al (2014) YAP/TAZ incorporation in 

the b-catenin destruction complex orchestrates the Wnt response. Cell 

158: 157 – 170 

22. Ying Q-L, Stavridis M, Griffiths D, Li M, Smith A (2003) Conversion of 

embryonic stem cells into neuroectodermal precursors in adherent 

monoculture. Nat Biotechnol 21: 183 – 186 

23. Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van der Kooy D (2001) 

Direct Neural Fate Specification from Embryonic Stem Cells. Neuron 30: 

65 – 78 

24. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang 

W, Marraffini LA et al (2013) Multiplex genome engineering using 

CRISPR/Cas systems. Science 339: 819 – 823 

25. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church 

GM (2013) RNA-guided human genome engineering via Cas9. Science 

339: 823 – 826 

26. Kim J, Woo AJ, Chu J, Snow JW, Fujiwara Y, Kim CG, Cantor AB, Orkin SH 

(2010) A Myc network accounts for similarities between embryonic stem 

and cancer cell transcription programs. Cell 143: 313 – 324 

27. Home P, Saha B, Ray S, Dutta D, Gunewardena S, Yoo B, Pal A, Vivian JL, 

Larson M, Petroff M et al (2012) Altered subcellular localization of transcription 

factor TEAD4 regulates first mammalian cell lineage commitment. 

Proc Natl Acad Sci USA 109: 7362 – 7367 

28. Zhao B, Ye X, Yu J, Li L, Li W, Li S, Yu J, Lin JD, Wang C-Y, Chinnaiyan 

AM et al (2008) TEAD mediates YAP-dependent gene induction and 

growth control. Genes Dev 22: 1962 – 1971 

29. Zhao B, Kim J, Ye X, Lai ZC, Guan KL (2009) Both TEAD-binding and 

WW domains are required for the growth stimulation and oncogenic 

transformation activity of yes-associated protein. Cancer Res 69: 

1089 – 1098 

30. Hailesellasse SK, Porter CJ, Palidwor G, Perez-Iratxeta C, Muro EM, 

Campbell PA, Rudnicki MA, Andrade-Navarro MA (2007) Gene function 

in early mouse embryonic stem cell differentiation. BMC Genom 8: 85 

31. Xiao JH, Davidson I, Matthes H, Garnier J-M, Chambon P (1991) Cloning, 

expression, and transcriptional properties of the human enhancer factor 

TEF-1. Cell 65: 551 – 568 

528 



32. Mahoney W, Hong J, Yaffe M, Farrance I (2005) The transcriptional coactivator 

TAZ interacts differentially with transcriptional enhancer 

factor-1 (TEF-1) family members. Biochem J 225: 217 – 225 

33. Farrance I, Mar J, Ordahl C (1992) M-CAT binding factor is related to the 

SV40 enhancer binding factor, TEF-1. J Biol Chem 267: 17234 – 17240 

34. Morin-Kensicki EM, Boone BN, Howell M, Stonebraker JR, Teed J, Alb JG, 

Magnuson TR, O’Neal W, Milgram SL (2006) Defects in yolk sac vasculogenesis, 

chorioallantoic fusion, and embryonic axis elongation in mice 

with targeted disruption of Yap65. Mol Cell Biol 26: 77 – 87 

35. Beyer TA, Weiss A, Khomchuk Y, Huang K, Ogunjimi AA, Varelas X, Wrana 

JL (2013) Switch enhancers interpret TGF-b and hippo signaling to 

control cell fate in human embryonic stem cells. Cell Rep 5: 1611 – 1624 

36. Amit M, Carpenter MK, Inokuma MS, Chiu C-P, Harris CP, Waknitz MA, 

Itskovitz-Eldor J, Thomson JA (2000) Clonally derived human embryonic 

stem cell lines maintain pluripotency and proliferative potential for 

prolonged periods of culture. Dev Biol 227: 271 – 278 

37. Nichols J, Chambers I, Taga T, Smith A (2001) Physiological rationale for 

responsiveness of mouse embryonic stem cells to gp130 cytokines. 

Development 128: 2333 – 2339 

38. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren 

MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and 

quantification by RNA-Seq reveals unannotated transcripts and 

isoform switching during cell differentiation. Nat Biotechnol 28: 

511 – 515 


529


Resistance of glia-like central and peripheral neural 

stem cells to genetically induced mitochondrial 

dysfunction—differential effects on neurogenesis 

Blanca Díaz-Castro 1,† , Ricardo Pardal 1,2,3,† , Paula García-Flores 1,3 , Verónica Sobrino 1 , Rocío Durán 1 , 

José I Piruat 1,** & José López-Barneo 1,2,3,* 

Abstract 

Mitochondria play a central role in stem cell homeostasis. Reversible 

switching between aerobic and anaerobic metabolism is critical 

for stem cell quiescence, multipotency, and differentiation, as 

well as for cell reprogramming. However, the effect of mitochondrial 

dysfunction on neural stem cell (NSC) function is unstudied. 

We have generated an animal model with homozygous deletion of 

the succinate dehydrogenase subunit D gene restricted to cells of 

glial fibrillary acidic protein lineage (hGFAP-SDHD mouse). Genetic 

mitochondrial damage did not alter the generation, maintenance, 

or multipotency of glia-like central NSCs. However, differentiation 

to neurons and oligodendrocytes (but not to astrocytes) was 

impaired and, hence, hGFAP-SDHD mice showed extensive brain 

atrophy. Peripheral neuronal populations were normal in hGFAP- 

SDHD mice, thus highlighting their non-glial (non hGFAP + ) lineage. 

An exception to this was the carotid body, an arterial chemoreceptor 

organ atrophied in hGFAP-SDHD mice. The carotid body 

contains glia-like adult stem cells, which, as for brain NSCs, are 

resistant to genetic mitochondrial damage. 

Keywords carotid body stem cells; mitochondrial dysfunction; neural stem 

cells; peripheral versus central neurogenesis 

Subject Categories Stem Cells; Neuroscience 

DOI 10.15252/embr.201540982 | Received 7 July 2015 | Revised 20 August 

2015 | Accepted 21 August 2015 | Published online 21 September 2015 

EMBO Reports (2015) 16: 1511–1519 

Introduction 

Intermediary metabolism plays a critical role in stem cell maintenance 

and differentiation. Somatic stem cells in their niches are 

normally in a quiescent state and maintained under a predominantly 

anaerobic condition, which helps to preserve them from 

excessive production of reactive oxygen species and other stressors 

[1,2]. Indeed, hypoxia is believed to be an essential environmental 

factor for maintenance of the stemness in embryonic and adult 

stem cells [3,4]. Upregulation of hypoxia-inducible glycolytic genes 

is a hallmark of somatic stem cells [5,6] as well as embryonic and 

induced pluripotent stem cells (iPSCs) [7]. In contrast, oxidative 

phosphorylation appears to be necessary for hematopoietic stem 

cell (HSC) differentiation to mature cells [8]. An inverse metabolic 

switch (i.e., involving a change from aerobic to anaerobic metabolism) 

has also been reported to occur upon reprogramming of 

adult cells to iPSCs [7,9]. Although the role of mitochondria in 

pluripotency and differentiation is a topic at the forefront of stem 

cell research [2,7], the actual effect of in vivo mitochondrial 

dysfunction on stem cell survival and differentiation is poorly 

understood. This is particularly evident in the case of neural stem 

cells (NSCs), despite the fact that oxidative phosphorylation is 

critical for neuronal homeostasis, possibly more than for any other 

cell type. 

Adult multipotent NSCs exist both in the central (CNS) and the 

peripheral (PNS) nervous system. During CNS development, radial 

glia, a cell type originated from the primordial neuroepithelium, 

serve as stem cells from which many neurons in cortical and subcortical 

areas as well as astrocytes and oligodendrocytes are derived 

[10,11]. A population of NSCs of astroglial lineage remains in the 

subventricular zone (SVZ) and the hippocampus of the adult 

mammalian brain, thereby supporting neurogenesis throughout 

life [11]. In contrast with the CNS, the PNS derives from a nonhomogeneous 

population of neural crest progenitors that migrate 

throughout the embryo and give rise to neurons and peripheral glia 

during fetal and early postnatal life [12,13]. Multipotent neural crest 

progenitor cells in the PNS also persist in adult life, particularly in 

the enteric nervous system (ENS) [14,15] and in the carotid body 

(CB), a neural crest-derived peripheral chemoreceptor organ located 

at the carotid artery bifurcation that grows in response to hypoxia 

[16,17]. Whether CB stem cells and other peripheral adult NSCs 

1 Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Seville, Spain 

2 Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, Sevilla, Spain 

3 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 

*Corresponding author. Tel: +34 955 923007; E-mail: lbarneo@us.es 

**Corresponding author. Tel: +34 955 923088; E-mail: jpiruat-ibis@us.es 

† These authors contributed equally to this work 


EMBO reports Mitochondrial dysfunction in neural stem cells Blanca Díaz-Castro et al 

share a similar glial phenotype and metabolic properties with 

central neurogenic progenitors is not known. 

We have generated a mouse model with conditional deletion of 

the gene encoding the membrane anchoring subunit D of succinate 

dehydrogenase restricted to cells expressing Cre recombinase under 

control of the human glial fibrillary acidic protein (hGFAP) 

promoter (hGFAP-SDHD mouse), which is active in mouse radial 

glia (see Materials and Methods). It is known that ablation of this 

mitochondrial complex II (MCII) gene in catecholaminergic cells 

compromises ATP synthesis and results in oxidative stress and 

neuronal loss in the brain and PNS [17,18]. This new animal model 

has permitted us to examine experimentally the resistance of neural 

stem cells to genetically induced mitochondrial dysfunction, which 

has remained untested so far. The experiments have also provided 

valuable information on the differential origin and properties of 

neural stem cells in the CNS and PNS, which might be relevant to 

their ability to support neurogenesis in adult life. 


Genetic modification of mitochondrial function in embryonic 

radial glia alters neuronal maturation during 

brain development 

Mutant (hGFAP-SDHD) mice were viable and did not exhibit any 

obvious alterations at birth. At postnatal day (P) 0, brains of 

mutant animals had similar appearance as those of control littermates, 

although dilation of ventricles was observed in some cases 

(Fig EV1). However, during the second week of life, hGFAP-SDHD 

mice exhibited a marked phenotype characterized by a lack of 

motor coordination, ataxia and decreased body size (Fig EV2A). 

Animals rapidly deteriorated and died between P16 and P18. At 

P15, the brains of GFAP-SDHD mice displayed notable malformations 

and were approximately 50% smaller than those of controls 

(Fig 1A). Anatomical and histological differences between brains 

of mutant mice and controls are illustrated in Fig 1B–M. Coronal 

sections stained with the neuronal marker NeuN at different 

rostro-caudal levels indicated marked atrophy of the cerebral 

cortex, particularly the dorso-lateral region, and a virtual absence 

of the hippocampus and cerebellum in hGFAP-SDHD mice with 

respect to controls, which at this age had already developed a 

normal adult brain. Detailed cytoarchitectonic or neuronal identification 

analyses were outside the scope of this work; however, the 

disappearance of cortical layers in the fronto-parietal region and 

atrophy of the corpus callosum were clearly evident histological 

hallmarks of hGFAP-SDHD mice (Fig 1B–E). In these animals, the 

regions normally occupied by the hippocampus and cerebellum 

contained only embryonic primordial rudiments of these structures 

(Fig 1F–M). The temporo-cortical areas, basal nuclei, and ventrocaudal 

brain (diencephalon and brain stem) appeared less affected 

(Fig 1B, C, F and G; see sagittal sections and details of structures 

at higher magnification in Fig EV2B–K). Staining of P0 and P15 

brains of wild-type and mutant mice with antibodies against GFAP 

showed in both cases the presence of numerous GFAP + cells 

(Fig EV3). Interestingly, glial cells in hGFAP-SDHD mice had a 

radial glia-like phenotype, with long processes, which were not 

present in normal GFAP + cells (Fig EV3H, I, K and L). Histological 

analyses at an intermediate (P5) stage of development are shown 

in Fig EV4. We established that the amount of SdhD functional 

allele was clearly diminished in P15 hGFAP-SDHD mice in comparison 

with homo- or heterozygous normal littermates (Fig 1N). 

MCII activity in brain mitochondria of hGFAP-SDHD mice was also 

decreased to less than 30% of that in homozygous controls 

(Fig 1O), thus confirming the deletion of the SdhD gene in a significant 

proportion of brain cells. In accord with the morphological 

data, these biochemical differences were not observed when only 

the ventral parts of the brains were used for analysis (data not 

shown). SdhD mRNA levels and MCII activity were already 

decreased in newborn (P0) hGFAP-SDHD brains, indicating that 

ablation of the SdhD gene had taken place during embryonic life, 

as soon as the hGFAP promoter became active (see Fig EV1M and 

N). To ensure that the Cre-mediated loxP recombination had actually 

resulted in deletion of the SdhD alleles in GFAP + cells, we 

generated control and hGFAP-SDHD mouse lines that expressed 

the enhanced green fluorescent protein (EGFP) driven by the 

hGFAP promoter (see Materials and Methods). After dissociation, 

GFAP + cells were sorted from two different brain areas (cortex 

and striatum) (Fig 1P and Q). In both cases, the levels of SdhD 

mRNA were reduced to < 20% of the value in control mice (Fig 1R 

and S), thus demonstrating that the SdhD gene was ablated in 

GFAP + cells from hGFAP-SDHD mice. These findings suggest that 

brain NSCs, along with their progeny of neuroblasts and astrocytes 

generated before birth, appear to be little affected by mitochondrial 

dysfunction. However, neuronal maturation, a process that occurs 

in the perinatal period, seems to be highly dependent on proper 

mitochondrial oxidative metabolic function. 

The extensive brain atrophy in hGFAP-SDHD mice is in fair 

agreement with previous lineage-specific tracing experiments showing 

that, during development, radial glia are stem cells from which 

many central neurons in the cortical and subcortical areas are 

derived [10]. Although these data, using Cre/loxP recombination of 

reporter genes, could be susceptible to misinterpretation due to the 

failure of reporter activity, our experiments, based on recombination 

of an intrinsic gene (SdhD) essential for cell homeostasis, fully confirm 

these results. Indeed, regional brain atrophy in the hGFAP- 

SDHD mice is almost superimposable on the distribution of labeled 

(X-gal + ) cells described in lineage tracing experiments using the 

same human GFAP promoter to drive Cre recombinase expression 

[10]. Relative preservation of ventral brain areas in hGFAP-SDHD 

animals can therefore be explained by the differential distribution of 

Cre-mediated recombination, which in hGFAP-Cre mice spares 

neural cells in this region. 

Adult SVZ stem cells are preserved in SDHD-deficient mice, but 

survival of newly generated neurons and oligodendrocytes 

is impaired 

Adult NSCs in the SVZ, which are considered to derive from radial 

glia, also have a GFAP + astrocyte-like phenotype [11]. We therefore 

investigated whether mitochondrial dysfunction influenced the 

maintenance, proliferation, and/or multipotency of these stem cells. 

Neurosphere assays of dispersed SVZ cells obtained from control 

and hGFAP-SDHD animals showed that the number of clonal colonies 

generated was the same for the two mouse strains (Fig 2A and B). 

Self-renewal capacity, as evidenced by the number of secondary 

1512 


Blanca Díaz-Castro et al Mitochondrial dysfunction in neural stem cells EMBO reports 

A B C D E 

N 

F G H 

I 

O 

J 

K 

L 

M 

P 

R 

S 

Q 

Figure 1. 


1513


◀ 

Figure 1. Brain structures and mitochondrial complex II activity in wild-type and hGFAP-SDHD mice. 

A Photographs of control (top) and hGFAP-SDHD (bottom) brains at P15. Scale bar: 5 mm. 

B–M Brain sections of postnatal (P15) control (B, D, F, H, J, L) and hGFAP-SDHD (C, E, G, I, K, M) mice immunostained with NeuN antibody. Scale bars: 1 mm (B, C, F, G, 

J, K), 500 lm (D, E, H, I), and 200 lm (L, M). Ctx: cortex; cc: corpus callosum; CPu: caudate putamen; hc: hippocampus; Dg: dentate gyrus. 

N Relative levels of functional SdhD allele in the entire brain (P15) as determined by qPCR of genomic DNA. r.u.: relative units (n = 4). 

O Succinate–ubiquinone oxidoreductase (SQR) activity of mitochondria isolated from the entire brain (P15) (n = 6). 

P, Q Representative profiles of sorted astrocytes from cortex and striatum of mice carrying the hGFAP-EGFP transgene (blue lines). Animals without the construct (black 

line) were used as blanks to determine the selection gates. 

R, S Relative SdhD mRNA levels in sorted GFAP-expressing astrocytes from the cortex and striatum of control and hGFAP-SDHD mice. 

Data information: Data are presented as mean SEM (n = 4–8). Statistical significance: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. The ANOVA test with the appropriate 

post hoc analysis was applied. See also Figs EV1–EV4. 

neurospheres, was also unaffected by the SdhD deletion (Fig EV5). 

These data suggest that SVZ stem cells are well preserved in 

hGFAP-SDHD mice with defective mitochondria. Nonetheless, the 

size of the primary (Fig 2A and C) and secondary (Fig EV5C) 

neurospheres was smaller in hGFAP-SDHD mice compared to 

controls (see below). Quantitative PCR analysis of SdhD mRNA 

from neurospheres confirmed the ablation of the SdhD gene in SVZ 

cells of genetically modified mice (Fig 2D). Furthermore, SVZ stem 

A B C D 

E 

F 

G 

H 

I 

Figure 2. Effects of mitochondrial dysfunction on subventricular zone neural stem cells. 

A Bright-field images of neurospheres obtained from SVZ neural stem cells of P15 control and hGFAP-SDHD mouse brains. Scale bars: 500 lm. 

B, C Neurosphere (NS) forming efficiency (B) and core diameter (C) in cultures grown from SVZ of P15 control and hGFAP-SDHD mice (n = 6 cultures/mice for each genotype). 

D Quantitative RT–PCR detection of SdhD expression levels in SVZ neurospheres of wild-type (flox/+) and mutant (flox/ , and flox/ cre) mice (n = 3–4 mice on each group). 

E Immunofluorescence detection of the neuronal marker Tuj1 in SVZ neural stem cell-derived adherent cultures from P15 control or hGFAP-SDHD brains. Nuclei were 

counterstained with DAPI. Scale bar: 25 lm. 

F Number of Tuj1 + neurons generated in SVZ neural stem cell adherent cultures in vitro for several days (n = 8 cultures/mice for each genotype). 

G Number of GFAP + astrocytes present in adherent cultures of SVZ neurospheres illustrating the resistance of glial cells to the loss of mitochondrial function (n = 5 

independent cultures). 

H Immunofluorescence detection of the oligodendrocyte marker O4 in SVZ neural stem cell-derived adherent cultures from P15 control or hGFAP-SDHD brains. Nuclei 

were counterstained with DAPI. Scale bar: 25 lm. 

I Number of O4 + neurons generated in SVZ neural stem cell adherent cultures in vitro for several days (n = 5–7 cultures/mice for each genotype). See also Fig EV5. 

Data information: Data are presented as mean SEM. *P ≤ 0.05. The two-tailed Student’s t-test was applied. 

1514 



cells from hGFAP-SDHD animals were able to differentiate into 

Tuj1 + neurons in vitro; however, survival of these newly generated 

neurons was compromised (Fig 2E and F). In contrast, the differentiation 

and survival of astrocytes were practically unaltered in SdhDdefective 

neurospheres (Fig 2E and G). Survival of differentiated 

oligodendrocytes was also decreased in preparations from hGFAP- 

SDHD mice (Fig 2H and I). These observations provide direct experimental 

support for the notion that, as other progenitor cell types 

[5,6], central NSCs rely predominantly on anaerobic metabolism and 

thus can survive and maintain their normal function even after 

severe mitochondrial damage. However, maintenance of mature 

neurons and oligodendrocytes, but not GFAP + astrocytes, is 

absolutely dependent on a correctly functioning mitochondrial 

metabolism. 

hGFAP-SDHD mice have normal PNS development but impaired 

carotid body neurogenesis 

Despite the gross brain developmental alterations exhibited by the 

hGFAP-SDHD mice, they showed an apparently normal PNS. The 

morphology and number of neurons in the adrenal medulla, superior 

cervical ganglion (SCG), ENS, and dorsal root ganglia, all of 

which are derived from neural crest progenitor cells [12], were similar 

in P15 hGFAP-SDHD mice compared with controls (Fig 3A–H). 

We checked that, as in the CNS glia, GFAP + Schwann cells in 

peripheral nerves were also lacking the SdhD alleles, thereby 

demonstrating recombination of the floxed SdhD alleles in PNS 

structures (Fig 3I and J). Interestingly, ablation of the SdhD gene 

did not seem to compromise survival of these peripheral glial cells 

(Fig 3K), although extensive loss of TH + adrenal chromaffin cells 

and SCG neurons has been observed in previous studies with TH + - 

specific SdhD-deficient (TH-SDHD) mice [18]. Quantitative PCR and 

histological analyses showed no differences in the SCG of hGFAP- 

SDHD and control mice (Fig EV6). A small, and non-significant, 

decrease in SdhD mRNA observed in the SCG of hGFAP-SDHD mice 

with respect to controls probably reflected SdhD deletion in the 

small population of GFAP + glial cells existing in this structure 

(Fig EV6A and B). Lack of hGFAP-Cre-dependent activity in peripheral 

neurons was further demonstrated in vivo using a LacZ reporter 

mice [16], which showed the absence of b-gal staining in TH + 

neurons from SCG (Fig EV6C and D). Taken together, these data 

suggest that NSCs giving rise to PNS neurons do not have the 

GFAP + glial origin that is characteristic of brain NSCs. An exception 

to this general rule appears to be the CB, which in newborn (P0) 

hGFAP-SDHD mice had similar size to that of control animals 

(Fig 4A–C). However, the normal postnatal increase in TH + glomus 

cell number and the volume of TH + CB parenchyma observed in 

controls were abolished in hGFAP-SDHD mice (Fig 4A–C). Despite 

the decrease in CB neuron-like glomus cell number, the population 

of CB stem cells in the hGFAP-SDHD mice remained unaltered as 

judged by the ability of dispersed CB stem cells to form growing 

neurospheres (Fig 4D–F). These observations indicate that unlike 

other structures in the PNS, postnatal development of the CB 

depends on glia-like GFAP + stem cells that are also resistant to 

mitochondrial dysfunction. A population of these glia-like cells 

remains dormant in the adult CB and sustains its adaptive growth 

upon exposure to chronic hypoxia [16,17]. Other stem cells in the 

adult PNS do not seem to contribute to the physiological homeostasis 

of the specific tissues where they are located [14,15]. Therefore, 

NSCs of glial lineage that reside in the adult CB constitute a 

neurogenic niche of similar characteristics to those existing in the 

adult brain and confer upon the CB the anatomical and functional 

plasticity required for acclimatization to hypoxia [17]. The expression 

of GFAP in brain and CB neural stem cells could in fact be the 

manifestation of a quiescent state in these cells that is necessary to 

maintain their capacity to generate neurons in adulthood. 

Mitochondria and neural stem cell proliferation 

and differentiation 

We have shown that low reliance on mitochondrial oxidative 

phosphorylation seems to be a common generic feature of NSCs 

during development and in the adult niches. Nonetheless, the way 

different stem cell classes achieve a homeostatic anaerobic metabolism 

may be variable. Several groups have reported a smaller 

number of mitochondria and modifications of their shape in multipotent 

HSCs in comparison to more-committed bone marrow 

progenitors [2]. Moreover, the high glycolytic flux of quiescent 

HSCs residing in hypoxic niches seems to rely on the upregulation 

of hypoxia-inducible transcription factors (particularly HIF-1a) and 

the subsequent induction of glycolytic enzymes and pyruvate 

dehydrogenase kinase to blunt pyruvate-dependent oxidative phosphorylation 

[5,6,19]. However, it has been reported that although 

stabilization of HIF-1a and HIF-2a is necessary to initiate the metabolic 

switch in the early stages of the reprogramming of human 

cells to iPSCs, the stabilization of HIF-2a during latter stages 

represses reprogramming [9]. HIF-1a-deficient NSCs have normal 

mitochondria although they are resistant to hypoxia and have high 

glycolytic activity [20]. Recently, we showed that maintenance and 

clonal growth of CB stem cells in vitro is unaffected by a broad 

range of O 2 tensions [17]. This insensitivity to O 2 tension is also 

observed in neurosphere cultures of adult SVZ stem cells [21] as 

well as in embryonic stem cells [3]. Although we have observed 

that survival of central (SVZ) and peripheral (CB) neural stem cells 

Figure 3. Normal development and survival of peripheral neurons in hGFAP-SDHD mice. 

A–H Immunofluorescence detection of neuronal markers TH (A, C) and HuC/D (E, G) in the adrenal medulla (A), superior cervical ganglion (C), enteric ganglia at the level of the 

distal small intestine (E), and dorsal root ganglion (G) of P15 control and hGFAP-SDHD mice. Scale bars: 200 lm (A, C), 100 lm (E, G). Cell number in adrenal medulla (B), 

superior cervical ganglion (D), enteric ganglia (F), and dorsal root ganglion (H) in the same animal models. Data are presented as mean SEM (n = 4–7 per group). 

I Immunofluorescence detection of GFAP expression in cross sections of peripheral sciatic nerve illustrating the normal shape of Schwann cells forming myelin 

sheaths in P15 control and hGFAP-SDHD mice. Scale bar: 50 lm. 

J Results of quantitative RT–PCR to detect SdhD expression in the peripheral nerves of wild-type (flox/+) and mutant (flox/ , and flox/ cre) mice. Data are presented 

as mean SEM (n = 3 flox/+, n = 6 flox/ , and n = 8 flox/ cre mice). Statistical significance: *P ≤ 0.05; **P ≤ 0.01. The two-tailed Student’s t-test was applied. 

K Number of myelin sheaths per unit area in sciatic nerves of P15 control and mutant mice. Data are presented as mean SEM (n = 4 mice for each genotype). See 

also Fig EV6. 

▸ 


1515


A 

B 

C 

D 

E 

F 

G 

H 

I J K 

Figure 3. 

1516 



A 

B 

D 

C 

E 

characteristic properties of stem cells regardless of O 2 tension 

levels. Therefore, besides the hypoxic stabilization of HIF, other 

intrinsic mechanisms and/or niche factors might contribute to the 

anaerobic metabolic state associated with the maintenance of NSC 

multipotency. 

Although mitochondrial dysfunction was not seen to alter NSC 

maintenance and multipotency in the present study, it exerted a 

marked effect on the survival of their central and peripheral neural 

progeny. Defective mitochondrial metabolism seemed to impair 

radial glial cell differentiation, as in P15 animals they appeared in a 

state typical of the prenatal brain. However, these mutated radial 

glia cells were able to differentiate into neurons, oligodendrocytes, 

and astrocytes. Brains from neonatal wild-type and hGFAP-SDHD 

mice showed similar sizes, structures, and NeuN + cell densities, 

suggesting that neuronal maturation occurring during the first postnatal 

weeks rather than neuroblast differentiation is the process that 

was actually compromised in mitochondria-defective animals. On 

the other hand, we observed the appearance of neurons and 

oligodendrocytes in differentiation assays of SVZ stem cells from 

hGFAP-SDHD mice, although these cells did not survive longer than 

a few days. Understanding the role of mitochondria on stemness 

and the switching from quiescence to activity in stem cells located 

in the different niches may help in the development of new therapeutic 

strategies against cancer stem cells. 


Figure 4. Impairment of carotid body postnatal maturation with 

maintenance of adult stem cells in hGFAP-SDHD mice. 

A Immunofluorescence detection of TH expression in newborn (P0) and P15 

wild-type (control) and hGFAP-SDHD mouse carotid body (CB). Boundaries of 

the CB parenchyma are indicated by the dotted lines. Scale bar: 50 lm. 

B, C Number of TH + cells (B) and size (C) per CB at different ages in control 

and hGFAP-SDHD mice. Data are presented as mean SEM (n = 3–5 

mice in each group). *P ≤ 0.05; **P ≤ 0.01. The two-tailed Student’s 

t-test was applied. 

D Bright-field images of CB neurospheres obtained from control and 

mutant mice at P15. Scale bar: 200 lm. 

E, F Neurosphere forming efficiency (E) and diameter (F) of floating cultures 

of dispersed CB cells from P15 control and hGFAP-SDHD mice. Data are 

presented as mean SEM (n = 6 cultures/mice for each genotype). 

is not affected by mitochondrial dysfunction, proliferation of SVZ 

progenitors (as estimated by the size of neurosphere cores) was 

reduced in preparations from hGFAP-SDHD mice. This could 

indicate that the high rate of in vitro proliferation of central 

progenitors (in comparison with progenitors in CB neurospheres) 

makes them partially dependent on energy obtained by oxidative 

phosphorylation. Taken together, these data suggest that a high 

glycolytic flux and resistance to mitochondrial dysfunction are 

F 

Animals 

The hGFAP-SDHD strain with SdhD flox/ hGFAP-CRE genotype was 

obtained by breeding the previously reported SdhD-flox mouse 

strain [18] with a mouse line expressing Cre under control of the 

human GFAP promoter, which is active in mouse radial glia 

[10,22]. Littermates with SdhD flox/+ and SdhD flox/ genotypes lacking 

CRE recombinase are referred to as flox/+ and flox/ , respectively. 

Where indicated, results from both genotypes were pooled 

and assigned to a control group since no differences between them 

were found for the phenotypes tested. Routine genotyping was 

performed for the SdhD alleles and the CRE gene by PCR as previously 

reported [10,22]. hGFAP-SDHD mice carrying the enhanced 

green fluorescence protein (EGFP) under control of the human 

GFAP promoter were obtained by breeding with the hGFAP-EGFP 

strain [23]. Mice were housed under temperature-controlled conditions 

(22°C) on a 12-h light/dark cycle, and provided with food 

and water ad libitum. Mice were housed and treated according to 

the animal care guidelines of the European Community Council 

(86/609/EEC), as well as institutional guidelines approved by the 

ethics committee of the Hospital Universitario Virgen del Rocio 

and the University of Seville (see Appendix Supplementary 

Materials and Methods). 

Histochemistry and immunocytochemistry 

For detection of NeuN, GFAP, TH, O4, HuC/D, Tuj1, and b-gal in 

tissue sections, neurospheres, and dissociated cells, we used standard 

staining procedures. Specific details are given in the Appendix 

Supplementary Materials and Methods. 


1517


Tissue dissociation, cell sorting, and neurosphere assays 

Dissociated cells were obtained from brain and CB tissue following 

standard procedures. Cell sorting and the generation of SVZ and CB 

neurospheres were carried out as indicated previously [16]. See 

Appendix Supplementary Materials and Methods. 

SdhD DNA and mRNA analyses 

DNA and RNA analyses were performed as described previously 

[18]. See Appendix Supplementary Materials and Methods. 

Mitochondria isolation and complex II activity 

Mitochondrial complex II activity was determined according to 

Piruat et al [24] with slight modifications. See Appendix Supplementary 

Materials and Methods. 

Statistics 

Data are presented as mean standard error (SEM). Statistical 

significance was assessed by ANOVA with appropriate post hoc 

analysis. For paired groups, either a two-tailed Student’s t-test with 

a Levene test for homogeneity of variances in the case of normal 

distribution, or the nonparametric Mann–Whitney U-test in the case 

of non-normal distributions, was applied. Normal distribution was 

assessed by Shapiro–Wilk test. PASW18 software was used for 

statistical analysis. 

Expanded View for this article is available online: 

http://embor.embopress.org 


This research is supported by grants from the Botín Foundation (JL-B), the 

Spanish Ministry of Science and Innovation SAF program (JL-B, RP and JIP), the 

European Research Council (RP and JL-B), and the Junta de Andalucía (JIP). We 

thank Alberto Castejón, Valentina Annese, and María José Castro for technical 

assistance. 


BD-C, RP, PG-F, VS, RD, and JIP performed experiments. BD-C, RP, JIP, and JL-B 

designed experiments and contributed to generate a draft of the manuscript. 

JL-B coordinated the project. 



References 

1. Suda T, Takubo K, Semenza GL (2011) Metabolic regulation of hematopoietic 

stem cells in the hypoxic niche. Cell Stem Cell 9: 298 – 310 

2. Xu X, Duan S, Yi F, Ocampo A, Liu GH, Izpisua Belmonte JC (2013) 

Mitochondrial regulation in pluripotent stem cells. Cell Metab 18: 

325 – 332 

3. Ezashi T, Das P, Roberts RM (2005) Low O 2 tensions and the prevention 

of differentiation of hES cells. Proc Natl Acad Sci USA 102: 

4783 – 4788 

4. Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A (2010) Oxygen in 

stem cell biology: a critical component of the stem cell niche. Cell Stem 

Cell 7: 150 – 161 

5. Simsek T, Kocabas F, Zheng J, Deberardinis RJ, Mahmoud AI, Olson EN, 

Schneider JW, Zhang CC, Sadek HA (2010) The distinct metabolic profile 

of hematopoietic stem cells reflects their location in a hypoxic niche. 

Cell Stem Cell 7: 380 – 390 

6. Takubo K, Nagamatsu G, Kobayashi CI, Nakamura-Ishizu A, Kobayashi 

H, Ikeda E, Goda N, Rahimi Y, Johnson RS, Soga T et al (2013) 

Regulation of glycolysis by Pdk functions as a metabolic checkpoint 

for cell cycle quiescence in hematopoietic stem cells. Cell Stem Cell 

12: 49 – 61 

7. Folmes CD, Nelson TJ, Dzeja PP, Terzic A (2012) Energy metabolism 

plasticity enables stemness programs. Ann N Y Acad Sci 1254: 82 – 89 

8. Inoue S, Noda S, Kashima K, Nakada K, Hayashi J, Miyoshi H (2010) 

Mitochondrial respiration defects modulate differentiation but not 

proliferation of hematopoietic stem and progenitor cells. FEBS Lett 584: 

3402 – 3409 

9. Mathieu J, Zhou W, Xing Y, Sperber H, Ferreccio A, Agoston Z, 

Kuppusamy KT, Moon RT, Ruohola-Baker H (2014) Hypoxia-inducible 

factors have distinct and stage-specific roles during reprogramming of 

human cells to pluripotency. Cell Stem Cell 14: 592 – 605 

10. Malatesta P, Hack MA, Hartfuss E, Kettenmann H, Klinkert W, Kirchhoff 

F, Gotz M (2003) Neuronal or glial progeny: regional differences in radial 

glia fate. Neuron 37: 751 – 764 

11. Kriegstein A, Alvarez-Buylla A (2009) The glial nature of embryonic and 

adult neural stem cells. Annu Rev Neurosci 32: 149 – 184 

12. Le Douarin N, Dulac C, Dupin E, Cameron-Curry P (1991) Glial cell 

lineages in the neural crest. Glia 4: 175 – 184 

13. Morrison SJ, White PM, Zock C, Anderson DJ (1999) Prospective 

identification, isolation by flow cytometry, and in vivo self-renewal 

of multipotent mammalian neural crest stem cells. Cell 96: 737 – 749 

14. Joseph NM, He S, Quintana E, Kim YG, Nunez G, Morrison SJ (2011) 

Enteric glia are multipotent in culture but primarily form glia in the 

adult rodent gut. J Clin Invest 121: 3398 – 3411 

15. Laranjeira C, Sandgren K, Kessaris N, Richardson W, Potocnik A, Vanden 

Berghe P, Pachnis V (2011) Glial cells in the mouse enteric nervous 

system can undergo neurogenesis in response to injury. J Clin Invest 

121: 3412 – 3424 

16. Pardal R, Ortega-Saenz P, Duran R, Lopez-Barneo J (2007) Glia-like stem 

cells sustain physiologic neurogenesis in the adult mammalian carotid 

body. Cell 131: 364 – 377 

17. Platero-Luengo A, Gonzalez-Granero S, Duran R, Diaz-Castro B, Piruat JI, 

Garcia-Verdugo JM, Pardal R, Lopez-Barneo J (2014) AnO 2 -sensitive 

glomus cell-stem cell synapse induces carotid body growth in chronic 

hypoxia. Cell 156: 291 – 303 

18. Diaz-Castro B, Pintado CO, Garcia-Flores P, Lopez-Barneo J, Piruat JI 

(2012) Differential impairment of catecholaminergic cell maturation and 

survival by genetic mitochondrial complex II dysfunction. Mol Cell Biol 

32: 3347 – 3357 

19. Romero-Moya D, Bueno C, Montes R, Navarro-Montero O, Iborra FJ, Lopez 

LC, Martin M, Menendez P (2013) Cord blood-derived CD34 + hematopoietic 

cells with low mitochondrial mass are enriched in hematopoietic 

repopulating stem cell function. Haematologica 98: 1022 – 1029 

20. Candelario KM, Shuttleworth CW, Cunningham LA (2013) Neural stem/ 

progenitor cells display a low requirement for oxidative metabolism 

independent of hypoxia inducible factor-1alpha expression. J Neurochem 

125: 420 – 429 

1518 



21. d’Anglemont de Tassigny X., Sirerol-Piquer M.S., Gómez-Pinedo U., Pardal 

R., Bonilla S., Capilla-Gonzalez V., López-López I., De la Torre-Laviana F.J., 

García-Verdugo J.M., López-Barneo J. (2015). Resistance of subventricular 

neural stem cells to chronic hypoxemia despite structural 

disorganization of the germinal center and impairment of neuronal and 

oligodendrocyte survival. Hypoxia 3: 15 – 33. 

22. Zhuo L, Theis M, Alvarez-Maya I, Brenner M, Willecke K, Messing A 

(2001) hGFAP-cre transgenic mice for manipulation of glial and 

neuronal function in vivo. Genesis 31: 85 – 94 

23. Nolte C, Matyash M, Pivneva T, Schipke CG, Ohlemeyer C, Hanisch UK, 

Kirchhoff F, Kettenmann H (2001) GFAP promoter-controlled EGFPexpressing 

transgenic mice: a tool to visualize astrocytes and astrogliosis 

in living brain tissue. Glia 33: 72 – 86 

24. Piruat JI, Pintado CO, Ortega-Saenz P, Roche M, Lopez-Barneo J (2004) 

The mitochondrial SDHD gene is required for early embryogenesis, and 

its partial deficiency results in persistent carotid body glomus cell 

activation with full responsiveness to hypoxia. Mol Cell Biol 24: 

10933 – 10940 


1519


UTX inhibits EMT-induced breast CSC properties by 

epigenetic repression of EMT genes in cooperation 

with LSD1 and HDAC1 

Hee-Joo Choi 1,† , Ji-Hye Park 2,† , Mikyung Park 3 , Hee-Young Won 1 , Hyeong-seok Joo 1 , Chang Hoon Lee 3 , 

Jeong-Yeon Lee 2,* & Gu Kong 1,2,** 

Abstract 

The histone H3K27 demethylase, UTX, is a known component of 

the H3K4 methyltransferase MLL complex, but its functional association 

with H3K4 methylation in human cancers remains largely 

unknown. Here we demonstrate that UTX loss induces epithelial– 

mesenchymal transition (EMT)-mediated breast cancer stem cell 

(CSC) properties by increasing the expression of the SNAIL, ZEB1 

and ZEB2 EMT transcription factors (EMT-TFs) and of the transcriptional 

repressor CDH1. UTX facilitates the epigenetic silencing of 

EMT-TFs by inducing competition between MLL4 and the H3K4 

demethylase LSD1. EMT-TF promoters are occupied by c-Myc and 

MLL4, and UTX recognizes these proteins, interrupting their transcriptional 

activation function. UTX decreases H3K4me2 and H3 

acetylation at these promoters by forming a transcriptional repressive 

complex with LSD1, HDAC1 and DNMT1. Taken together, our 

findings indicate that UTX is a prominent tumour suppressor that 

functions as a negative regulator of EMT-induced CSC-like properties 

by epigenetically repressing EMT-TFs. 

Keywords breast CSC; EMT; UTX 

Subject Categories Chromatin, Epigenetics, Genomics & Functional 

Genomics; Cancer 

DOI 10.15252/embr.201540244 | Received 13 February 2015 | Revised 10 July 

2015 | Accepted 14 July 2015 | Published online 24 August 2015 

EMBO Reports (2015) 16: 1288–1298 

Introduction 

A ubiquitously transcribed tetratricopeptide repeat X chromosome 

(UTX) functions as a histone demethylase towards di- and tri-methylated 

histone H3 on lysine 27 (H3K27me2/H3K27me3) [1]. This 

demethylase is also a known component of the mixed-lineage 

leukaemia (MLL) 2/3 H3K4 methyltransferase complex, although its 

catalytic effect on H3K4me remains unclear [2–4]. Polycomb group 

(PcG)-dependent H3K27 methylation has redundant functions in 

many biological processes, including stem cell regulation and 

tumour development [5–7], although less evidence supports 

the functional role of UTX in these cellular processes. Several 

studies suggest that UTX regulates somatic cell reprogramming and 

embryonic development [8,9], as well as tumour suppression in 

leukaemia [10,11]. Furthermore, genomewide analyses have identified 

various somatic mutations and deletions of UTX in several 

human cancers, including breast, bladder, and renal cancers and 

leukaemia [12–14]. However, the demethylation-dependent or 

demethylation-independent molecular function of UTX in human 

cancers has not been clearly explained, and it remains controversial 

whether UTX has tumour-suppressive activity [15–17]. 

Accumulating evidence shows that many histone methyltransferases 

and demethylases are involved in the regulation of 

cancer stem cells (CSCs), a small population of stem-like cancer 

cells possessing tumorigenic and metastatic capacities [18,19]. 

Moreover, recent evidence suggests that regulators of the epithelial– 

mesenchymal transition (EMT), which repress E-cadherin (encoded 

by CDH1) transcriptionally, such as SNAIL, SLUG, TWIST, ZEB1 

and ZEB2, play a key role in inducing CSC self-renewal [20–23]. 

Notably, these EMT transcription factors (EMT-TFs) cooperate with 

various chromatin-modifying enzymes (including the histone 

methylases G9a and Set8 and the H3K4/K9 demethylase LSD1) as 

well as histone deacetylases and DNMTs to form a repressive 

complex for CDH1 [24–26]. UTX is also known to interact physically 

with transcriptional and epigenetic regulators, such as MLL 2/3 

H3K4 methyltransferase [2–4], histone acetyltransferase and the 

SWI/SNF complex [27,28], during the assembly of multi-protein 

complexes. However, the molecular relationship between UTX and 

EMT-TFs in the epigenetic regulation of EMT-associated CSC properties 

remains unknown. 

Herein, we found that UTX is critical for inhibiting EMT-induced 

breast CSC properties by suppressing the c-Myc-dependent 

1 Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea 

2 Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, Korea 

3 College of Pharmacy, Dongguk University, Seoul, Korea 

*Corresponding author. Tel: +82 2 2220 0634; Fax: +82 2 2295 1091; E-mail: jy2jy2@hanyang.ac.kr 

**Corresponding author. Tel: +82 2 2290 8251; Fax: +82 2 2295 1091; E-mail: gkong@hanyang.ac.kr 


1288 


Hee-Joo Choi et al Repression of EMT-induced CSC by UTX EMBO reports 

transcription of SNAIL, ZEB1 and ZEB2 in cooperation with H3K4 

demethylase LSD1, HDAC1 and DNMT1. These findings suggest a 

novel epigenetic mechanism for UTX during its inhibition of EMT- 

TFs as a tumour suppressor in human breast cancer. 


UTX loss enhances CSC-like properties and the EMT in human 

breast cancer 

Given the evidence that Drosophila and murine Utx antagonize 

Notch signalling and activate tumour suppressor Rb proteins, previous 

studies have suggested a putative tumour-suppressive function 

for UTX in human cancer [15,16]. Consistently, in human breast 

cancer, UTX somatic mutations have been discovered that might be 

associated with the loss of UTX function [12]. In contrast, a recent 

study suggested that UTX has oncogenic potential in human breast 

cancer cell lines [17]. To clearly determine the role of UTX in 

human cancer, we evaluated the expression of UTX in several cell 

lines derived from several stages of breast tumour development. 

UTX expression was downregulated in the cancer cell lines 

compared with normal or immortalized cells (Fig 1A). Notably, 

UTX mRNA was expressed at lower levels in CD44 + /CD24 /ESA + 

cells with stem cell-like characteristics (Fig 1B). Thus, we investigated 

whether UTX affects stem-like phenotypes in normal and 

malignant breast epithelial cells. The percentage of CD44 + /CD24 / 

ESA + cells in the cell population was increased by UTX knockdown 

in the immortalized MCF10A cells and in breast cancer cell lines 

highly expressing UTX; however, this population declined in 

response to UTX overexpression in MDA-MB-231 cells having a low 

UTX expression (Figs 1C and EV1A and B). UTX also negatively 

regulated mammosphere formation and anchorage-independent 

growth (Fig 1D and E). Consistently, in vivo breast tumour xenograft 

assays indicated that mice bearing UTX-overexpressing MDA- 

MB-231 tumours exhibited retarded tumour initiation and growth 

(Table EV1, Fig EV1C), whereas mice injected with UTX-deficient 

SK-BR-3 cells presented more rapid tumour formation and growth 

than control mice (Table EV1, Fig EV1D). Moreover, UTX knockdown 

induced the neoplastic transformation of the non-tumorigenic 

MCF10A cell lines in vivo (Fig EV1E). Considering the ability of 

CD44 + /CD24 /ESA + cells to transform mammary epithelial cells 

[22,23], these data suggest that UTX loss might promote breast 

tumorigenesis by expanding stem-like cells and enhancing their selfrenewal 

and tumour-initiating capacities. 

Recent studies have shown that EMT is essential for generating 

CD44 + /CD24 low/ stem-like cells that can convert breast epithelial 

cells into tumorigenic cells and can confer breast cancer aggressiveness 

[20–23]. Consistent with these findings, UTX knockdown in 

MCF10A cells resulted in a mesenchymal-like sporadic long spindle 

phenotype (Fig 2A). We also observed a reduced expression of 

epithelial markers including E-cadherin but an increased expression 

of mesenchymal markers in these cells (Fig 2B–D). Furthermore, 

UTX-overexpressing MDA-MB-231 cells exhibited epithelial-like 

morphology (Fig 2A), increased epithelial marker expression and 

decreased mesenchymal marker expression (Fig 2B–D). Contrary to 

a recent study showing decreased invasion after UTX knockdown in 

breast cancer [17], EMT promotion resulting from UTX loss 

conferred invasion and migration abilities on non-invasive MCF10A 

cells, whereas UTX overexpression inhibited these effects in invasive 

MDA-MB-231 cells (Fig 2E and F). Consistently, a meta-analysis 

of breast cancer patient survival performed using the Kaplan–Meier 

Plotter [29] showed that high UTX expression is associated with 

favourable overall survival (P < 0.001, log-rank test; n = 741), 

relapse-free survival (P

EMBO reports Repression of EMT-induced CSC by UTX Hee-Joo Choi et al 

A 

B 

C 

D 

E 

Figure 1. Loss of UTX enhances CSC-like properties in human breast cancer. 

A Screening of protein expression levels by immunoblotting in normal and cancerous breast cell lines. 

B UTX mRNA expression in CD44 + /CD24 /ESA + cells (CSC) and other cells (non-CSC) was sorted using flow cytometry from the indicated cell lines and analysed using 

qRT-PCR. ***P < 0.001 versus Non-CSC; two-tailed unpaired t-test. 

C Flow cytometry analysis was used to measure CD44 + /CD24 /ESA + cell populations in the indicated cell lines. The lower right quadrant indicates the CD44 + /CD24 / 

ESA + cells (x-axis, APC-conjugated CD44; y-axis, PE-conjugated CD24). CON si, non-targeting siRNA; UTX si, UTX siRNA; CON, pLVX-puro empty vector; UTX, pLVXpuro-UTX. 

*P < 0.05, **P < 0.01, ***P < 0.001 versus controls; two-tailed unpaired t-test. 

D, E Mammosphere formation (D) and soft agar colony formation (E) in cells that underexpress or overexpress UTX. After 21 days (E) or as indicated (D), the numbers of 

spheres (D) or colonies (E) (> 100 lm diameter) were counted. shCON, control shRNA; shUTX, UTX shRNA. *P < 0.05, **P < 0.01, ***P < 0.001 versus controls; 

two-tailed unpaired t-test. 

Data information: The data shown in (B–E) represent the means SD of n = 3 independent experiments. 

Source data are available online for this figure. 

1290 



A 

B 

C 

D 

E 

F 

Figure 2. 

Loss of UTX induces the EMT and invasion of breast cancer cells. 

A Representative bright-field images of cells that underexpress or overexpress UTX. Scale bars: 100 lm. 

B, C The expression of epithelial and mesenchymal markers in the indicated cells was measured using immunoblotting (B) and qRT-PCR (C). 

D The immunofluorescence staining of E-cadherin (red) and vimentin (green) was detected using confocal microscopy and quantified. DAPI (blue) was used for 

nuclear staining. Scale bars: 10 lm. 

E, F Chamber transwell assays of cellular invasion or migration by the indicated cells. Invasion and migration of test cells are expressed relative to values for control 

cells. Scale bars: 100 lm. 

Data information: Error bars in (C–F) indicate the SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 versus controls; two-tailed unpaired t-test. 



1291


A 

B 

C 

D 

E 

F 

G 

Figure 3. 

1292 



◀ 

Figure 3. UTX depletion inhibits E-cadherin transcription by recruiting EMT-related transcription factors. 

A To measure CDH1 promoter activity, a luciferase (luc) assay was performed in UTX-knockdown MCF10A and in UTX-overexpressing MDA-MB-231 cells that were cotransfected 

with either CDH1 wild-type (WT) or E-box mutant (Mut)-luc and b-galactosidase constructs for 24 h. 

B, C The expression of EMT-TFs was measured in the indicated cells using immunoblotting (B) and qRT-PCR (C). 

D Immunoblotting with cytoplasmic and nuclear fractions to analyse EMT-TF expression. SP1 and a-tubulin were used for loading controls for nuclear and 

cytoplasmic proteins, respectively. 

E Immunofluorescence analysis showing the expression level and cellular localization of SNAIL, ZEB1 and ZEB2 in UTX-knockdown MCF10A cells. Nuclei were stained 

with DAPI. Scale bar: 10 lm. 

F ChIP analysis showing the indicated histone methylation status and gene recruitment to the CDH1 promoter in UTX-deficient MCF10A cells. IgG, control IgG for 

negative control. 

G Flow cytometry analysis of the CD44 + /CD24 /ESA + population in MCF10A cells that were transfected with non-targeting siRNA (CON si) or with UTX-targeting 

siRNA (UTX si), together with the indicated EMT-TF siRNAs for 48 h. 

Data information: The data shown in (A, C, E, F and G) represent the means SD of n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus controls; 

†† P < 0.01 versus UTX si; ns, no significance (two-tailed unpaired t-test for A, C, E and F; one-way ANOVA and Scheffe’s post hoc test for G). 


these conditions (Fig EV2E and F). Although H3K27me3 was 

decreased by EZH2 knockdown in the CDH1 promoter, recruitment 

of EMT-TFs to this region was maintained continuously in the UTXknockdown 

cells (Fig EV2G). Therefore, these data imply that UTX 

regulates EMT and stemness in an EZH2 activity-independent 

manner; this finding supports UTX as a crucial inhibitor of EMTassociated 

breast tumorigenesis that might overcome the function of 

EZH2 in breast cancer. 

UTX transcriptionally represses EMT-TF expression by altering 

c-Myc-dependent and c-Myc-independent 

epigenetic modifications 

Little is known about the epigenetic and transcriptional mechanisms 

that occur in the genomic regions of EMT-TFs; in contrast, 

the CDH1 regulatory mechanism has been well established. Therefore, 

we next investigated the molecular mechanism by which UTX 

transcriptionally regulates EMT-TFs to inhibit the properties of 

EMT-induced CSCs. SNAIL, ZEB1 and ZEB2 were found to have 

E-box motifs that bind c-Myc in their proximal promoters; therefore, 

we examined the effect of UTX on c-Myc activity towards EMT- 

TFs. Although the expression of c-Myc was not altered by UTX 

(Fig EV3A), the recruitment of c-Myc to these regions was 

enhanced by UTX knockdown in MCF10A cells and was inhibited 

by UTX overexpression in MDA-MB-231 cells, as assessed using a 

ChIP assay (Figs 4A and EV3B–D). In addition, UTX bound directly 

to these regions. Previous studies have shown that many UTXoccupied 

genes not only exhibit altered H3K27 methylation states 

but also altered H3K4 methylation states [2,3,31], although the 

mechanism by which H3K4 methylation is regulated via UTX 

remains unclear. To identify whether UTX-associated histone modifications 

participate in the transcriptional regulation of EMT-TFs, 

we examined the modification status of histone proteins including 

H3K27me3 and H3K4me2 at these promoters. Although the level of 

H3K27me3 remained unchanged, the H3K4me2 and H3 acetylation 

(ac) active histone markers were enriched in the SNAIL, ZEB1 and 

ZEB2 promoters in UTX-deficient MCF10A cells. Moreover, the 

recruitment of p300 histone acetyltransferase (HAT) and the dissociation 

of HDAC1 and DNMT1 transcriptional repressive markers 

were observed in these regions. Consistent results were found in 

UTX-overexpressing MDA-MB-231 cells. Furthermore, co-immunoprecipitation 

(co-IP) of exogenous and endogenous proteins 

showed that UTX physically interacts with c-Myc, HDAC1 and 

DNMT1 (Fig 4B and C). To determine whether c-Myc acts as a 

bridge for the recognition of UTX by the target promoter and consequent 

epigenetic changes, we analysed epigenetic changes at the 

EMT-TF promoters following siRNA-mediated c-Myc depletion. 

Although c-Myc was required for changes in H3ac by UTX, it did 

not affect the enrichment of UTX and H3K4me2 in these promoters 

(Fig EV4A and B). However, flow cytometry following c-Myc 

siRNA treatment showed that c-Myc depletion reverses UTX-knockdown-induced 

stem cell expansion (Fig 4D). Taken together, these 

results show that UTX represses the transcription of EMT-TFs by 

inhibiting c-Myc-dependent histone acetylation and inducing 

c-Myc-independent H3K4 demethylation. Together with the results 

of a previous study showing the regulation of SNAIL by c-Myc [32], 

our data suggest that c-Myc is a common transcription factor for the 

induction of SNAIL, ZEB1 and ZEB2 expression through its role in 

coordinating with epigenetic modifiers that involve an active gene 

state. Furthermore, UTX might be a key component for the epigenetic 

silencing of these EMT-TFs to antagonize c-Myc function. 

UTX cooperates with LSD1 demethylase and inhibits MLLmediated 

H3K4me to epigenetically repress EMT-TFs 

To further explain how UTX negatively regulates the H3K4me2 level 

at the EMT-TF promoters in a c-Myc-independent manner, we investigated 

possible histone modification enzymes targeting H3K4me 

that might cooperate with UTX in regulating EMT-TFs. Consistent 

with previous findings [2,4,17], we observed that UTX physically 

interacts with the H3K4-specific methyltransferase MLL4 (Fig 5A). 

Interestingly, UTX also bound H3K4 demethylase LSD1 and 

enhanced the association between LSD1 and MLL4, as confirmed by 

co-IP results. Furthermore, ChIP analysis revealed that MLL4 consistently 

bound to the EMT-TF promoters regardless of UTX expression, 

whereas LSD1 recruitment to these regions occurred only in 

the presence of UTX expression in both UTX-overexpressing and 

UTX-knockdown cells, thereby suggesting UTX-dependent LSD1 

recruitment and H3K4 demethylation at the EMT-TF promoters 

(Fig 5B). These data also imply that UTX is not a binding partner of 

MLL4 but rather inhibits H3K4 methylation by facilitating competition 

between MLL4 and LSD1. 

LSD1 role as an oncogene has been well described, despite 

controversy regarding in its role in breast cancer [33–35]. To define 

the potential role of LSD1 in the UTX-mediated repression of EMT- 

TFs, we inhibited LSD1 expression using lentiviral shRNA in 


1293


A 

B 

C 

D 

Figure 4. 

UTX suppresses EMT-TF expression by inducing c-Myc-dependent and c-Myc-independent epigenetic modification. 

A ChIP analysis showing the histone methylation status and recruitment of the indicated proteins within the EMT-TF promoters. *P < 0.05, **P < 0.01, ***P < 0.001 

versus controls (two-tailed unpaired t-test). 

B, C The interaction between UTX and the indicated proteins was confirmed by co-IP in 293 T cells transfected with the indicated constructs (B) and in UTXoverexpressing 

MDA-MB-231 cells (C). 

D MCF10A cells were transfected with CON si or UTX si, together with c-Myc si. The CD44 + /CD24 /ESA + population was then evaluated using flow cytometry. 

**P < 0.01 versus CON si; †† P < 0.01 versus UTX si (one-way ANOVA and Scheffe’s post hoc test). 

Data information: The results shown in (A and D) represent the means SD of three independent experiments. 


1294 



A 

B 

C 

D 

E 

Figure 5. 


1295


◀ 

Figure 5. UTX cooperates with LSD1 demethylase to inhibit H3K4me in EMT-TF promoters. 

A The binding of UTX to the indicated proteins was analysed using co-IP experiments involving lysates from 293T cells transfected with the indicated constructs. 

B ChIP analysis showing the recruitment of LSD1 and MLL4 to the EMT-TF promoters. *P < 0.05, **P < 0.01, ***P < 0.001 versus controls (two-tailed unpaired 

t-test). 

C, D UTX-overexpressing MDA-MB-231 cells were infected with LSD1 shRNA viruses; immunoblotting (C) and ChIP analyses (D) were then performed to confirm the 

expression of the indicated proteins and the epigenetic changes at the SNAIL promoters. *P < 0.05, **P < 0.01, ***P < 0.001 versus. CON/shCON; † P < 0.05, 

†† P < 0.01, ††† P < 0.001 versus UTX/shCON (one-way ANOVA and Scheffe’s post hoc test for c-Myc IP; Welch’s test and Dunnett’s T3 post hoc test for others). 

E Schematic summary of the regulation of EMT-TFs by UTX. 

Data information: The data shown in (B and D) represent the means SD of n = 3 independent experiments. 


control or UTX-overexpressing MDA-MB-231 cells. Consistent with 

previous findings that LSD1 promotes EMT [35,36], we found that 

LSD1 knockdown decreased SNAIL and ZEB1 expression and 

increased E-cadherin expression in control MDA-MB-231 cells 

expressing low levels of UTX (Fig 5C). In contrast, the loss of LSD1 

recovered the expression of EMT-TFs that were inhibited by UTX 

and subsequently abolished E-cadherin expression (Fig 5C), indicating 

that UTX overexpression causes LSD1 to function as a negative 

regulator of EMT-TFs. These paradoxical results support the 

critical role of UTX in determining the oncogenic potential of LSD1 

in human breast cancer. Because LSD1 is a component of the 

CoREST and NuRD transcriptional repressive complexes, which 

contain HDACs [33,37], we assumed that UTX might recruit not 

only LSD1 but also LSD1-containing multiple repressive complexes 

to its target chromatin. Indeed, the ChIP analysis results showed 

that the repression of H3K4me2 and H3ac by the recruitment of 

LSD1 and HDAC1 was recovered in UTX-overexpressing cells by 

LSD1 knockdown (Figs 5D and EV4C and D), implying that LSD1 is 

required for the formation of a transcriptional repressive complex 

with UTX and HDAC1 at the EMT-TF promoters. However, LSD1 

could not alter the inhibitory effect of UTX on c-Myc binding to 

these promoters. Collectively, our results indicate that UTX epigenetically 

inhibits the transcription of EMT-TFs by cooperating 

with LSD1/HDAC1 in competition with c-Myc/MLL4 in the 

regulation of EMT and CSCs. Contrary to the findings of Kim and 

colleagues [17], these data suggest that UTX might utilize MLL4 as 

a recognition signal for chromatin binding but inhibits MLL4 activity 

towards H3K4 by recruiting the LSD1 demethylase-containing 

transcriptional repressive complex to silence the expression of 

EMT-TFs in breast cancer. 

In this study, we demonstrated that UTX plays an important 

role in breast tumour suppression as an epigenetic regulator of 

EMT-TFs. UTX negatively regulated EMT-induced breast CSC properties 

by transcriptionally suppressing SNAIL, ZEB1 and ZEB2 in 

an H3K27 demethylation activity-independent manner (Fig 5E). In 

the absence of UTX, EMT-TFs are transcriptionally activated by 

recruiting c-Myc/p300 to the EMT-TF promoters. MLL4, the H3K4 

methyltransferase, was occupied and methylated H3K4 in these 

regions. When UTX was present, the recruitment of c-Myc and 

p300 was disrupted, and LSD1/HDAC1/DNMT1 recognized the 

target region by promoting UTX binding to MLL4. The UTXcontaining 

repressive complex inhibits the H3K4me and H3ac, 

resulting in gene silencing of the EMT-TFs. Collectively, these findings 

suggest that UTX is a novel epigenetic silencer of EMT-TFs 

that represses EMT-associated breast CSC-like properties in an 

H3K27 methylation-independent manner by cooperating with H3K4 

demethylase LSD1 and HDAC1. 


Flow cytometry 

Breast CSC populations were isolated using flow cytometry as 

described previously [38], based on the expression of surface markers 

(CD44 + /CD24 /ESA + ). To evaluate UTX expression in CSC and 

non-CSC populations from breast cell lines, cell populations were 

divided, stained using the appropriate antibodies and collected; 

the cells were then analysed using a FACSAria flow cytometer (BD 

Biosciences, Franklin Lakes, NJ, USA). 

Mammosphere formation 

MCF10A cells (1 × 10 4 cells/well), T-47D cells (5 × 10 3 cells/well), 

SK-BR-3 cells (1 × 10 4 cells/well) and MDA-MB-231 cells (5 × 10 3 

cells/well) were cultured under previously described conditions 

[38]. After 3, 5 and 7 days, mammosphere formation was analysed 

and quantified using an inverted microscope. 

ChIP–qPCR assay 

ChIP assays were performed using a ChIP assay kit according to the 

manufacturer’s instructions (Upstate Biotechnology, Lake Placid, 

NY, USA). ChIP signal enrichment was determined using real-time 

quantitative PCR (qPCR) (signal/input ratio) conducted using the 

7300 Real-Time PCR System and the SYBR Green Master Mix 

(Applied Biosystems, Foster City, CA, USA). 

Statistical analysis 

The statistical significance of differences between the control and 

experimental groups was analysed using the two-tailed unpaired 

t-test after confirming that data were normally distributed based on 

the Shapiro–Wilk test as implemented in the SPSS (version 12.0; SPSS 

Inc., Chicago, IL, USA). The Levene’s test was used to verify equality 

of variances. For multiple comparisons, one-way ANOVA followed 

by Scheffe’s post hoc test (for equal variances) or Welch’s test with 

Dunnett’s T3 post hoc test (for unequal variances) was performed. To 

estimate the frequency of the tumour-initiating ability, limiting 

dilution assay results were calculated using L-Calc software. 

P-values < 0.05 were considered to indicate statistical significance. 

For further methods, see Appendix Supplementary Methods. 

Expanded View for this article is available online: 

http://embor.embopress.org 

1296 




This study was supported by the National Research Foundation of Korea 

(NRF), which is funded by the Korean Government (No. 2010-0020879). 


GK and J-YL designed and supervised the project. H-JC, J-YL and GK wrote the 

manuscript. H-JC and J-HP performed the experiments, analysed and organized 

the data. MP, H-YW, H-SJ and C-HL conducted the experiments and 

analysed the data. 



References 

1. Agger K, Cloos PA, Christensen J, Pasini D, Rose S, Rappsilber J, Issaeva I, 

Canaani E, Salcini AE, Helin K (2007) UTX and JMJD3 are histone H3K27 

demethylases involved in HOX gene regulation and development. Nature 

449: 731 – 734 

2. Issaeva I, Zonis Y, Rozovskaia T, Orlovsky K, Croce CM, Nakamura T, 

Mazo A, Eisenbach L, Canaani E (2007) Knockdown of ALR (MLL2) reveals 

ALR target genes and leads to alterations in cell adhesion and growth. 

Mol Cell Biol 27: 1889 – 1903 

3. Lee MG, Villa R, Trojer P, Norman J, Yan KP, Reinberg D, Di Croce L, 

Shiekhattar R (2007) Demethylation of H3K27 regulates polycomb 

recruitment and H2A ubiquitination. Science 318: 447 – 450 

4. Cho YW, Hong T, Hong S, Guo H, Yu H, Kim D, Guszczynski T, Dressler GR, 

Copeland TD, Kalkum M et al (2007) PTIP associates with MLL3- and 

MLL4-containing histone H3 lysine 4 methyltransferase complex. J Biol 

Chem 282: 20395 – 20406 

5. Richly H, Aloia L, Di Croce L (2011) Roles of the Polycomb group proteins 

in stem cells and cancer. Cell Death Dis 2: e204 

6. Cao Q, Yu J, Dhanasekaran SM, Kim JH, Mani RS, Tomlins SA, 

Mehra R, Laxman B, Cao X, Yu J et al (2008) Repression of E-cadherin 

by the polycomb group protein EZH2 in cancer. Oncogene 27: 

7274 – 7284 

7. Chang CJ, Yang JY, Xia W, Chen CT, Xie X, Chao CH, Woodward WA, 

Hsu JM, Hortobagyi GN, Hung MC (2011) EZH2 promotes expansion of 

breast tumor initiating cells through activation of RAF1-beta-catenin 

signaling. Cancer Cell 19: 86 – 100 

8. Wang C, Lee JE, Cho YW, Xiao Y, Jin Q, Liu C, Ge K (2012) UTX regulates 

mesoderm differentiation of embryonic stem cells independent of H3K27 

demethylase activity. Proc Natl Acad Sci USA 109: 15324 – 15329 

9. Mansour AA, Gafni O, Weinberger L, Zviran A, Ayyash M, Rais Y, Krupalnik 

V, Zerbib M, Amann-Zalcenstein D, Maza I et al (2012) The H3K27 

demethylase Utx regulates somatic and germ cell epigenetic reprogramming. 

Nature 488: 409 – 413 

10. Ntziachristos P, Tsirigos A, Welstead GG, Trimarchi T, Bakogianni S, Xu L, 

Loizou E, Holmfeldt L, Strikoudis A, King B et al (2014) Contrasting roles 

of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia. 

Nature 514: 513 – 517 

11. Van der Meulen J, Sanghvi V, Mavrakis K, Durinck K, Fang F, Matthijssens F, 

Rondou P, Rosen M, Pieters T, Vandenberghe P et al (2015) The 

H3K27me3 demethylase UTX is a gender-specific tumor suppressor in 

T-cell acute lymphoblastic leukemia. Blood 125: 13 – 21 

12. van Haaften G, Dalgliesh GL, Davies H, Chen L, Bignell G, Greenman C, 

Edkins S, Hardy C, O’Meara S, Teague J et al (2009) Somatic mutations 

of the histone H3K27 demethylase gene UTX in human cancer. Nat 

Genet 41: 521 – 523 

13. Gui Y, Guo G, Huang Y, Hu X, Tang A, Gao S, Wu R, Chen C, Li X, Zhou L 

et al (2011) Frequent mutations of chromatin remodeling genes in transitional 

cell carcinoma of the bladder. Nat Genet 43: 875 – 878 

14. Dalgliesh GL, Furge K, Greenman C, Chen L, Bignell G, Butler A, Davies H, 

Edkins S, Hardy C, Latimer C et al (2010) Systematic sequencing of renal 

carcinoma reveals inactivation of histone modifying genes. Nature 463: 

360 – 363 

15. Herz HM, Madden LD, Chen Z, Bolduc C, Buff E, Gupta R, Davuluri R, 

Shilatifard A, Hariharan IK, Bergmann A (2010) The H3K27me3 demethylase 

dUTX is a suppressor of Notch- and Rb-dependent tumors in Drosophila. 

Mol Cell Biol 30: 2485 – 2497 

16. Terashima M, Ishimura A, Yoshida M, Suzuki Y, Sugano S, Suzuki T (2010) 

The tumor suppressor Rb and its related Rbl2 genes are regulated by Utx 

histone demethylase. Biochem Biophys Res Commun 399: 238 – 244 

17. Kim JH, Sharma A, Dhar SS, Lee SH, Gu B, Chan CH, Lin HK, Lee MG 

(2014) UTX and MLL4 coordinately regulate transcriptional programs for 

cell proliferation and invasiveness in breast cancer cells. Cancer Res 74: 

1705 – 1717 

18. Visvader JE (2011) Cells of origin in cancer. Nature 469: 314 – 322 

19. Tam WL, Weinberg RA (2013) The epigenetics of epithelial-mesenchymal 

plasticity in cancer. Nat Med 19: 1438 – 1449 

20. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, 

Reinhard F, Zhang CC, Shipitsin M et al (2008) The epithelial-mesenchymal 

transition generates cells with properties of stem cells. Cell 133: 

704 – 715 

21. Scheel C, Weinberg RA (2012) Cancer stem cells and epithelialmesenchymal 

transition: concepts and molecular links. Semin Cancer 

Biol 22: 396 – 403 

22. Lim S, Becker A, Zimmer A, Lu J, Buettner R, Kirfel J (2013) SNAI1- 

mediated epithelial-mesenchymal transition confers chemoresistance 

and cellular plasticity by regulating genes involved in cell death and 

stem cell maintenance. PLoS ONE 8: e66558 

23. Hollier BG, Tinnirello AA, Werden SJ, Evans KW, Taube JH, Sarkar TR, 

Sphyris N, Shariati M, Kumar SV, Battula VL et al (2013) FOXC2 expression 

links epithelial-mesenchymal transition and stem cell properties in 

breast cancer. Cancer Res 73: 1981 – 1992 

24. Lin Y, Wu Y, Li J, Dong C, Ye X, Chi YI, Evers BM, Zhou BP (2010) The 

SNAG domain of Snail1 functions as a molecular hook for recruiting 

lysine-specific demethylase 1. EMBO J 29: 1803 – 1816 

25. Dong C, Wu Y, Yao J, Wang Y, Yu Y, Rychahou PG, Evers BM, Zhou BP 

(2012) G9a interacts with Snail and is critical for Snail-mediated 

E-cadherin repression in human breast cancer. J Clin Invest 122: 

1469 – 1486 

26. Yang F, Sun L, Li Q, Han X, Lei L, Zhang H, Shang Y (2012) SET8 

promotes epithelial-mesenchymal transition and confers TWIST dual 

transcriptional activities. EMBO J 31: 110 – 123 

27. Tie F, Banerjee R, Conrad PA, Scacheri PC, Harte PJ (2012) Histone 

demethylase UTX and chromatin remodeler BRM bind directly to CBP and 

modulate acetylation of histone H3 lysine 27. Mol Cell Biol 32: 2323 – 2334 

28. Miller SA, Mohn SE, Weinmann AS (2010) Jmjd3 and UTX play a 

demethylase-independent role in chromatin remodeling to regulate 

T-box family member-dependent gene expression. Mol Cell 40: 594 – 605 

29. Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, Szallasi Z 

(2010) An online survival analysis tool to rapidly assess the effect of 

22,277 genes on breast cancer prognosis using microarray data of 1,809 

patients. Breast Cancer Res Treat 123: 725 – 731 


1297


30. De Craene B, Berx G (2013) Regulatory networks defining EMT 

during cancer initiation and progression. Nat Rev Cancer 13: 

97 – 110 

31. Wang JK, Tsai MC, Poulin G, Adler AS, Chen S, Liu H, Shi Y, Chang HY 

(2010) The histone demethylase UTX enables RB-dependent cell fate 

control. Genes Dev 24: 327 – 332 

32. Smith AP, Verrecchia A, Faga G, Doni M, Perna D, Martinato F, Guccione 

E, Amati B (2009) A positive role for Myc in TGFbeta-induced Snail transcription 

and epithelial-to-mesenchymal transition. Oncogene 28: 

422 – 430 

33. Wang Y, Zhang H, Chen Y, Sun Y, Yang F, Yu W, Liang J, Sun L, 

Yang X, Shi L et al (2009) LSD1 is a subunit of the NuRD complex 

and targets the metastasis programs in breast cancer. Cell 138: 

660 – 672 

34. Lim S, Janzer A, Becker A, Zimmer A, Schule R, Buettner R, Kirfel J (2010) 

Lysine-specific demethylase 1 (LSD1) is highly expressed in ER-negative 

breast cancers and a biomarker predicting aggressive biology. Carcinogenesis 

31: 512 – 520 

35. Lin T, Ponn A, Hu X, Law BK, Lu J (2010) Requirement of the histone 

demethylase LSD1 in Snai1-mediated transcriptional repression during 

epithelial-mesenchymal transition. Oncogene 29: 4896 – 4904 

36. Ferrari-Amorotti G, Fragliasso V, Esteki R, Prudente Z, Soliera AR, 

Cattelani S, Manzotti G, Grisendi G, Dominici M, Pieraccioli M et al 

(2013) Inhibiting interactions of lysine demethylase LSD1 with snail/slug 

blocks cancer cell invasion. Cancer Res 73: 235 – 245 

37. Lee MG, Wynder C, Cooch N, Shiekhattar R (2005) An essential role for 

CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature 437: 

432 – 435 

38. Won HY, Lee JY, Shin DH, Park JH, Nam JS, Kim HC, Kong G (2012) Loss 

of Mel-18 enhances breast cancer stem cell activity and tumorigenicity 

through activating Notch signaling mediated by the Wnt/TCF pathway. 

FASEB J 26: 5002 – 5013 

1298 


Review 

“Histones and Chromatin” Review Series 

Chromatin remodeling and bivalent histone 

modifications in embryonic stem cells 

Arigela Harikumar & Eran Meshorer * 

Abstract 

Pluripotent embryonic stem cells (ESCs) are characterized by 

distinct epigenetic features including a relative enrichment of 

histone modifications related to active chromatin. Among these is 

tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands 

of the H3K4me3-enriched promoters in pluripotent cells also 

contain a repressive histone mark, namely H3K27me3, a situation 

referred to as “bivalency”. While bivalent promoters are not unique 

to pluripotent cells, they are relatively enriched in these cell types, 

largely marking developmental and lineage-specific genes which 

are silent but poised for immediate action. The H3K4me3 and 

H3K27me3 modifications are catalyzed by lysine methyltransferases 

which are usually found within, although not entirely 

limited to, the Trithorax group (TrxG) and Polycomb group (PcG) 

protein complexes, respectively, but these do not provide selective 

bivalent specificity. Recent studies highlight the family of ATPdependent 

chromatin remodeling proteins as regulators of bivalent 

domains. Here, we discuss bivalency in general, describe the 

machineries that catalyze bivalent chromatin domains, and portray 

the emerging connection between bivalency and the action of different 

families of chromatin remodelers, namely INO80, esBAF, and 

NuRD, in pluripotent cells. We posit that chromatin remodeling 

proteins may enable “bivalent specificity”, often selectively acting 

on, or selectively depleted from, bivalent domains. 

Keywords chromatin; chromatin remodeling; embryonic stem cells; epigenetics; 

histone modifications 

DOI 10.15252/embr.201541011 | Received 13 July 2015 | Revised 1 October 

2015 | Accepted 5 October 2015 | Published online 9 November 2015 

EMBO Reports (2015) 16: 1609–1619 

See the Glossary for abbreviations used in this article. 

Introduction 

The genetic information of a living cell is stored within the DNA. 

However, additional layers of regulation provide the epigenetic 

information, which, in concert with transcription factors, enables 

the same primary DNA sequence to confer different identities to 

different cell types, developmental stages, disease states, etc. 

In eukaryotes, the DNA is wrapped around a histone octamer 

comprised of a pair of each of the core histones H2A, H2B, H3, and 

H4, which together form the nucleosome. Nucleosomes are the 

basic repeating units of chromatin, and they are arranged in a higher 

order chromatin structure through the binding of linker histones, H1 

proteins, between adjacent nucleosomes. Thus, despite having the 

same genetic makeup, different transcriptional outcomes of different 

cell types of the same organism are achieved through a variety of 

epigenetic modifications including DNA methylation, histone posttranslational 

modifications (PTMs), chromatin organization, etc. So far, 

several histone modifications with physiological importance have 

been identified, such as acetylation, methylation, phosphorylation, 

ubiquitylation, sumoylation, ADP ribosylation, deimination, proline 

isomerization, biotinylation, citrullination, and more [1–3]. Apart 

from influencing local chromatin structure, these modifications are 

also recognized by specific adaptor proteins which in turn recruit 

protein complexes and thereby affect gene regulation. Some of these 

histone marks such as H3K4me3, H3K9ac, and H3K14ac are associated 

with actively transcribed genes and some other modifications, 

for example, H3K27me3 and H3K9me3, are enriched within 

repressed regions. Activation and repression are believed to occur, 

at least partly, through charge-mediated chromatin decompaction 

and chromodomain-containing protein binding, respectively [3,4]. 

Interestingly, a subset of promoters associated with both activating 

(H3K4me3) and repressive (H3K27me3) marks, also known as 

“bivalent” modifications, has been discovered in mouse embryonic 

stem cells (ESCs) [5,6]. Several recent reviews thoroughly covered 

the field of bivalency, especially in pluripotent ESCs [7–10]. Here, 

we focus on the emerging link between bivalent histone modifications 

and ATP-dependent chromatin remodeling in ESCs. The 

term “chromatin remodeling” is often used to describe any change 

or modification to chromatin including histone modification. Here, 

by “chromatin remodeling”, we specifically refer to the action of the 

family of ATP-dependent chromatin remodeling factors, described 

below. When other forms of chromatin structure alterations are 

referred to, we describe the specific mode of alteration or modification. 

We will briefly summarize initial and recent experiments 

establishing the existence and role of bivalent domains in undifferentiated 

ESCs and during differentiation, and will argue that both 

H3K4me3 and H3K27me3, and especially the cross talk between 

them, are intimately linked with chromatin remodeling in pluripotent 

ESCs. 

Department of Genetics, Institute of Life Sciences and The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 

*Corresponding author. Tel: +972 2 6585161; E-mail: meshorer@huji.ac.il 


EMBO reports Chromatin remodeling, bivalent histone marks, and ESCs Arigela Harikumar & Eran Meshorer 

Glossary 

ASH2L Ash2 (absent, small, or homeotic)-like (Drosophila) 

protein 

BAF250a AT-rich interactive domain 1A (Swi1 like) protein 

Bmi1 B lymphoma Mo-MLV insertion region 1 protein 

BRG1 SWI/SNF-related, matrix-associated, actin-dependent 

regulator of chromatin, subfamily a, member 4 

Cbx7 chromobox homolog 7 protein 

CFP1 CXXC finger 1 (PHD domain) protein 

CHARGE coloboma, heart defect, atresia choanae (also known as 

choanal atresia), retarded growth and development, 

genital abnormality, and ear abnormality 

CHD chromodomain-helicase-DNA-binding protein 

ChIP chromatin immunoprecipitation 

EED embryonic ectoderm development protein 

esBAF brahma-associated factor complex associated with 

embryonic stem cells 

ESC embryonic stem cell 

EZH enhancer of zeste homolog protein 

H3K27me3 trimethylated lysine-27 on histone H3 

H3K4me3 trimethylated lysine-4 on histone H3 

HCNE highly conserved non-coding elements 

INO80 inositol-requiring 80 

iPSC induced pluripotent stem cell 

ISWI imitation switch 

LSD lysine-specific demethylase 1 

MLL mixed lineage leukemia protein 

MNase micrococcal nuclease 

MYC v-myc avian myelocytomatosis viral oncogene homolog 

NuRD nucleosome remodeling deacetylase 

OCT4 POU domain, class 5, transcription factor 1 protein 

PcG polycomb group 

Phc polyhomeotic-like 1 (Drosophila) protein 

PRC polycomb repressive complex 

PTMs posttranslational modifications 

RBBP5 retinoblastoma-binding protein 5 

RING1B ring finger protein 2 

rRNA ribosomal RNA 

SET SET domain containing protein 

SMARCD1 SWI/SNF-related, matrix-associated, actin-dependent 

regulator of chromatin, subfamily d, member 1 protein 

SUZ12 suppressor of zeste 12 homolog (Drosophila) protein 

SWI/SNF SWItch/Sucrose non-fermentable 

Tip60 K(lysine) acetyltransferase 5 

TrxG trithorax group 

TSS transcription start site 

WDR5 WD repeat domain 5 protein 

Bivalent modifications 

The first evidence for the existence of bivalent modifications came 

from studies in pluripotent mouse ESCs [5,6]. Using sequential 

chromatin immunoprecipitation (ChIP) and tiling arrays, highly 

conserved non-coding elements (HCNEs) were found to be 

enriched with bivalent histone modifications H3K4me3 and 

H3K27me3, marking lowly expressed developmental regulators 

[6]. Shortly thereafter, early replicating genes were similarly 

shown to possess bivalent domains [5]. Depletion of the EED 

subunit of the Polycomb repressive complex 2 (PRC2) led to an 

almost complete loss of H3K27me3 resulting in an upregulation of 

the bivalent genes analyzed. Despite the presence of the activating 

histone marks, the expression of the bivalent genes in both studies 

varied from very low to no expression, suggesting that these 

genes are poised for immediate activation. Supporting this notion, 

upon differentiation, some of these bivalent modifications were 

resolved, either losing the H3K27me3 mark permitting their 

expression, or losing the H3K4me3, rendering them stably silent 

(Fig 1). 

Bivalent histone modifications were also identified in human 

ESCs [11,12] and induced pluripotent stem cells (iPSCs) [13–15], 

marking developmentally regulated genes, similar to the situation 

found in mESCs. Other stem cell types, such as hematopoietic stem 

cells, were also shown to possess a similar bivalent chromatin architecture, 

containing thousands of bivalently marked, developmentally 

regulated promoters [16]. However, although many of these 

bivalent domains are resolved during differentiation, a subset of 

promoters retains its bivalent state following even terminal differentiation 

[17]. Therefore, bivalency may not merely reflect a transient, 

flexible chromatin state during differentiation, but rather a condition 

present in most or even all cell types. Supporting this idea, bivalent 

domains were found in a number of different non-stem-cell lines 

[14,18], including differentiated human T cells, where weakly 

expressed genes were found to possess additional acetylation marks 

on H3K9 and H3K14 along with H3K4me3 and H3K27me3 in their 

promoters [19,20]. Bivalent domains were reported in several 

cancer cells as well [21–24], where they were suggested to promote 

their plasticity and responsiveness, potentially serving as a novel 

unexplored therapeutic avenue [22]. These studies suggest that 

bivalent modifications are present in both pluripotent and nonpluripotent 

cells, where they maintain genes largely in a repressed 

state, but at the same time, keep them poised for activation until a 

proper signal is perceived. 

Despite the rapidly expanding literature reporting different 

aspects of bivalent chromatin, whether bivalent domains serve an 

actual function has recently been questioned [25,26]. In addition, 

the existence of bivalent domains on the same nucleosome has not 

been unequivocally demonstrated. Apart from the many ChIP experiments, 

bivalent chromatin was shown using micrococcal nuclease 

(MNase) digestion of chromatin followed by liquid chromatography 

and mass spectrometry [27], suggesting an asymmetric configuration 

of chromatin on opposite H3 tails. However, all evidence to 

date relies on population studies, and therefore, the seeming presence 

of two opposing marks on the same nucleosome may be the 

outcome of cellular heterogeneity. Single nucleosome resolution, the 

smoking gun of bivalent chromatin, has yet to be reported. 

Establishment and maintenance of bivalent modifications 

in ESCs 

Trithorax group (TrxG) proteins and Polycomb group (PcG) proteins 

assemble into multimeric protein complexes and are largely, 

although not exclusively, responsible for the deposition of H3K4me3 

and H3K27me3 marks, respectively (Fig 2). The exact mechanism 

behind the recruitment of these protein complexes to specific sites is 

not entirely clear; however, initial studies showed that bivalent 

domains are predominantly associated with CpG islands in ESCs 

[6]. In addition to DNA methylation, multiple studies have demonstrated 

that selected histone modifications, several transcription 

factors (TFs), and some non-coding RNAs (ncRNAs) also play a role 

in this process [7]. 

1610 


Arigela Harikumar & Eran Meshorer Chromatin remodeling, bivalent histone marks, and ESCs EMBO reports 

BIVALENT GENE 

SILENCED GENE 

H3K27me3 

H3K27me3 

ACTIVATED GENE 

Figure 1. The bivalency concept. 

A bivalent gene, depicted as a boat (top left), is ready to go (sail up: H3K4me3) but is held in check (anchor: H3K27me3). Once the sail is down (top right), the gene is stably 

silenced (only H3K27me3), but if instead the anchor is lifted (bottom), the gene is promptly activated (only H3K4me3). [Correction added on 2 December 2015 after first online 

publication: “H4K4me3” has been corrected to “H3K4me3”.] 

H3K4me3 

In mammalian systems, SETD1A, SETD1B, and MLL complexes, 

among others, which share several subunits including WDR5, 

RBBP5, ASH2L, and DPY-30, catalyze the deposition of the 

H3K4me3 mark [28] (Fig 2). SETD1A/B complexes seem to be 

responsible for global H3K4me3 deposition, whereas MLL1–MLL4 

complexes likely serve more specific functions. Among those, 

MLL2, but not MLL1 or SETD1, was shown to act as the main 

methyltransferase at bivalent promoters [25,26]. MLL1 and MLL2 

contain DNA-binding domains termed CXXC or zinc finger CXXC 

(ZF-CXXC) motifs, which specifically recognize unmethylated CpG 

islands [29,30]. These motifs act to recruit MLL complexes to chromatin 

templates by promoting target site recognition. Similarly, 

SETD1A/B complexes contain a CXXC finger protein 1 (CFP1) 

subunit, which includes a DNA-binding domain selectively recognizing 

unmethylated CpGs [31]. Loss of CFP1 most strongly affects 

H3K4 methylation at promoters of highly expressed genes in ESCs, 

but not at bivalent gene promoters [32]. Other SETD1A/B and MLL 

components were also shown to be important in ESCs or early ESC 

differentiation. Knockdown of WDR5 or ASH2L, for instance, results 

in aberrant expression programs and defective self-renewal and 

pluripotency [33–36], and knockdown of RBBP5 or DPY-30 has little 

effect on self-renewal but leads to improper ESC neuronal differentiation 

[37]. Interestingly, knockdown of DPY-30 alters H3K4 methylation 

specifically at bivalent domains in ESCs [38], suggesting a 

selective developmentally related function of this subunit. MLL2 

depletion also results in skewed differentiation, along all three germ 

layers [39], and Mll2-null mice die before embryonic day E11.5, 

showing drastically reduced expression of several Hox genes [40]. 

Taken together, these studies highlight the important role that H3K4 

methylation and its maintenance plays in development, pluripotency, 

ESC biology, and early ESC differentiation. 

H3K27me3 

The PRC2 complex is responsible for the deposition of H3K27me3 

marks at bivalent promoters (Fig 2). The core PRC2 complex is 

composed of enhancer of zeste (EZH2 or EZH1), embryonic ectoderm 

development (EED), suppressor of zeste 12 (SUZ12), as well as 

RBBP4 (RbAp48) and RBBP7 (RbAp46) [41]. EZH2 is the catalytic 

subunit, acting as the methyltransferase of H3K27, which in turn is 

recognized by chromodomain-containing proteins such as CBX 

proteins, as well as by the EED subunit itself [42]. In mouse ESCs, 

CBX7, for instance, the primary CBX protein expressed in ESCs 

[43,44], was shown to interact with H3K27me3 thereby recruiting 


1611


WDR5 

Hcf1 

Menin 

SETD1A/B 

MLL1,3,4 

ASH2 

MLL2 

MLL2 

complex 

DPY30 

RBBP4 

H3K4me3 

EED 

PRC2 

EZH2 

SUZ12 

PRC2 

complex 

RBBP4 

RBBP7 

H3K27me3 

modifications and pluripotency, the exact role that pluripotency 

factors play at bivalent domains remains largely unclear. 

Depletion of individual subunits EED or SUZ12 results in deregulation 

of lineage-specific genes, although with minimal impact on 

cell viability and self-renewal [56–59], suggesting that PcG 

complexes serve little function in ESCs or that in ESCs, alternative 

compensatory mechanisms exist. In contrast to the situation, when 

ESCs are kept in an undifferentiated state, ESCs deficient of PRC2 

components exhibit aberrant differentiation potential when differentiation 

is induced [56–59]. These situations parallel the postimplantation 

lethality phenotypes observed in PRC2 knockout mouse 

models [60–62]. Concomitantly, depletion of PRC1 components 

such as RING1B and BMI1 also impairs proper differentiation 

[63–66]. Simultaneous depletion of RING1B and EED in ESCs 

provokes an even stronger inclination toward differentiation, 

although self-renewal can still be preserved under careful culture 

conditions, and prolonged differentiation results in cell death [59]. 

Taken together, these knockout models demonstrate that akin to 

TrxG proteins, PcG proteins—arguably through the control of 

bivalent target genes encoding developmental regulators—are vital 

for proper ESC differentiation. 

Figure 2. Main protein complexes catalyzing bivalent chromatin marks. 

Left: Protein complexes catalyzing H3K4 methylation (green flag). Right: The 

PRC2 complex catalyzing H3K27 methylation (red flag). Shown are only the main 

proteins and protein complexes catalyzing H3K4/H3K27 methylation. Less 

abundant subunits are not depicted. [Correction added on 2 December 2015 after 

first online publication: “H4K4” has been corrected to “H3K4”.] 

the PRC1 complex [43,45]. RING1B and BMI1 subunits of the PRC1 

complex in turn deposit the ubiquitination of H2AK119 [46,47]. It 

was further suggested that the deposition of PRC1 and the 

H2AK119ub mark (H2A ubiquitylated on lysine 119) are involved in 

RNA polymerase 2 (RNAPII) pausing in gene bodies, rendering their 

repression [48,49]. PRC1-related H2AK119ub1 was also shown to 

recruit PRC2 to chromatin, demonstrating a functional link between 

the two complexes [50]. Indeed, many developmentally regulated 

genes are marked with bivalent domains consisting of both PRC1 

and PRC2 complexes, but interestingly, a subset of these bivalent 

domains have been shown to be exclusively bound by PRC2 [51]. 

Unlike the PRC1/PRC2 double positive bivalent domains, these 

PRC2-specific bivalent domains usually decorate promoters of genes 

which are not bona fide developmental genes (often encoding for 

membrane proteins or proteins of unknown functions) and are only 

weakly conserved. These findings suggest an additional mechanism 

of silencing. In ESCs, pluripotency factor binding sites often coincide 

with positioning of core subunits of MLL and PRC2 complexes on 

bivalent domains [6,34,52]. In addition, key pluripotency components, 

such as OCT4 and MYC, have been shown to interact with 

components of the MLL and PRC protein complexes [53,54], 

suggesting a tight co-regulation between bivalent domains and the 

pluripotency network. Indeed, depletion of OCT4 in ESCs results in 

a selective reduction of H3K4me3 levels on selected genes, providing 

evidence for the tight relationship between the pluripotency 

network and H3K4me3 levels [34], although whether reduced 

H3K4me3 levels are a cause or consequence of reduced transcription 

is still under question [55]. While these observations suggest a 

functional connection between the maintenance of bivalent 

Chromatin remodeling and bivalency 

Accumulating evidence suggests a tight interplay between ATPdependent 

chromatin remodeling proteins and bivalent histone 

modifications. While this connection likely plays a role in most, if 

not all, cell types, it is particularly pertinent for pluripotent stem 

cells, because of the relative abundance of chromatin remodelers in 

ESCs [67], and because of their special connection with bivalency, 

as described below. 

Chromatin remodeling proteins are ATP-dependent complexes 

that usually contain a catalytic ATPase subunit in addition to regulatory 

factors mediating protein–protein and chromatin–protein interactions. 

Chromatin remodelers act to alter chromatin structure by 

several different mechanisms, including incorporation or ejection of 

histone octamers, sliding nucleosomes along chromatin templates, 

and, by histone exchange, altering nucleosome composition [68] 

(Fig 3). Chromatin remodeling proteins are generally divided into 

four major families: SWI/SNF (switch/sucrose non-fermentable), 

CHD (chromodomain-helicase DNA-binding), ISWI (imitation 

switch), and INO80 (inositol-requiring 80), each involving different 

protein complexes and often different and even opposing actions. A 

growing number of chromatin remodeling proteins have been linked 

to ESC function and pluripotency and were shown to play essential 

roles in stemness and/or early differentiation and development [69]. 

Interestingly, at least one member of each of these remodeler families 

was shown to be essential for early mouse development, before 

or during implantation [69], demonstrating the importance of chromatin 

remodelers in pluripotency and early fate decisions. 

One of the first clues to the involvement of chromatin remodeling 

proteins in regulating either H3K4 or H3K27 methylation in developmental 

genes came from an RNAi screen of chromatin-related 

proteins in mouse ESCs [70]. Among the dozens of proteins that 

were identified to have a potential role in maintaining the undifferentiated 

state in ESCs, the authors specifically identified seven 

subunits of the Tip60–p400 complex of the INO80 family. Using 

1612 



INSERTION 

EVICTION 

DIMER EXCHANGE 

SLIDING 

Figure 3. The classical actions of chromatin remodeling proteins. 

Shown are different functional outcomes mediated by ATP-dependent chromatin remodeling proteins, including nucleosome insertion (top left), nucleosome eviction (top 

right), dimer exchange (bottom left), and nucleosome sliding (bottom right). The chromatin remodeling proteins themselves are not depicted. 

biochemical and functional assays, the authors found that Tip60– 

p400 significantly co-localizes with H3K4me3, especially around the 

transcription start site (TSS), and that it mostly acts to repress gene 

expression in ESCs. Knockdown of Tip–p400 resulted in 4% deregulated 

genes, most of which were upregulated. Interestingly, many of 

the upregulated genes were found to be classical bivalent early differentiation 

genes, which are normally silent in ESCs, reminiscent of 

depletion of PcG components in ESCs [70]. 

Additionally, PcG proteins were also shown to be functionally 

linked to chromatin remodeling proteins in ESCs. This relationship 

was revealed following knockdown (KD) studies of the core component 

of the SWI/SNF esBAF complex, BRG1, in mouse ESCs [71]. 

Expression analysis following BRG1 KD revealed increased transcription 

of several PcG subunits of both PRC1 and PRC2 complexes 

including Bmi1, Cbx7, Ring1, Phc1, and Phc2, promoters of which 

were directly bound by BRG1, as revealed by ChIP-seq experiments 

[71,72]. Moreover, a PRC2 component required for ESC differentiation, 

Jarid2, interacts with esBAF [73], counteracting PRC2’s 

methyltransferase activity [74,75]. These results suggest a direct 

association of chromatin remodeling proteins both with promoters 

of PcG-related genes and with the PRC2 protein complex itself. 

However, no co-localization of PRC2 components with BRG1 was 

found at chromatin on a genomewide scale [71], hinting that their 

association is not required for chromatin binding. When BRG1 was 

knocked out in ESCs, global H3K27me3 levels were not altered, but 

H3K27me3 displayed a selective elevation in BRG1-activated genes 

and a decrease in BRG1-repressed genes [76]. This indicates that 

BRG1 directly regulates the level and distribution of H3K27me3 at 

its target genes. 

The strong connection between SWI/SNF chromatin remodeling 

proteins and H3K27me3 in ESCs was recently further supported by 

our own studies [77]. Screening for proteins that are differentially 

associated with chromatin between undifferentiated and differentiated 

ESCs, we identified the chromatin remodeling protein 

SMARCD1 (BAF60a), an additional component of the esBAF 

complex. ChIP-seq maps of SMARCD1 in ESCs revealed a distribution 

not dissimilar from that of H3K27me3 around transcription start 

sites (TSS) and a significant enrichment in promoters of bivalent 

genes. Analyzing genomewide maps of H3K27me3 and H3K4me3 

before and after SMARCD1 depletion revealed significant redistribution 

of these marks. In undifferentiated ESCs, both H3K4me3 and 

H3K27me3 were slightly elevated around TSSs upon SMARCD1 KD. 

In contrast, in differentiating ESCs, H3K4me3 was still elevated 

around TSSs, but H3K27me3 was dramatically reduced, by more 

than 75% around TSSs. Interestingly, bivalent genes were relatively 

protected from this wave of H3K27me3 elimination, indicating selective 

regulation of bivalent genes, by an unknown mechanism. Once 

again, no apparent changes in global levels were observed, as seen 

in the BRG1-KO ESCs. 

Regulation of bivalent histone modifications by the esBAF 

complex was further established by an inducible knockout system 

of the esBAF component BAF250a in mouse ESCs [78]. Mapping 

of nucleosomes, bivalent histone modifications, and the PcG 

component SUZ12 before and after BAF250a depletion demonstrated 

that BAF250a mediates nucleosome occupancy and 

H3K27me3 levels at the upregulated, but not the downregulated 

genes in ESCs, and that it exerts its function likely by regulating 

esBAF and PRC2. BAF250a KO led to an increase in nucleosome 

positioning and a decrease in H3K27me3 levels, especially in bivalent 

and developmental gene promoters in ESCs. These alterations, 

accompanied by aberrant expression of developmental and 

pluripotency genes, resulted in differentiation defects in the 

BAF250a KO ESCs [78]. This study supports a synergic role for 

esBAF and PRC2 in ESCs. 


1613


As indicated above, the catalytic subunit of the esBAF complex is 

BRG1. In human ESCs, BRG1 depletion resulted in elevated levels of 

the enhancer-associated histone modification H3K27ac at BRG1 

target gene enhancers [79]. This suggests that in addition to its function 

in mediating H3K27 methylation levels at promoters, BRG1 

may also act as a selective repressor at enhancer regions. Support 

for a dual role for BRG1 in regulating H3K27ac enhancer regions on 

one hand, and in maintaining PcG-mediated promoter repression on 

the other, came from a recent study of in vitro mesodermal differentiation 

of mouse ESCs [80]. BRG1 co-localization with H3K27ac at 

distal enhancers is required to maintain their H3K27 acetylation 

during mesoderm induction, while it is also required to maintain 

PcG-mediated repression of non-mesodermal developmental regulators 

during differentiation. Taken together, these studies demonstrate 

a potential role for esBAF chromatin remodeling proteins in 

regulating bivalent histone modifications, primarily H3K27 methylation, 

in ESCs and during ESC differentiation, and provide evidence 

that the interplay between esBAF and PcG acts both to activate and 

to silence gene expression programs in ESCs. 

Another family of chromatin remodeling proteins which was implicated 

in regulating H3K4/H3K27 methylation is the Chromodomainhelicase 

DNA-binding (CHD) family of proteins. CHD1 was found 

to regulate open chromatin and pluripotency in mouse ESCs by 

its association with H3K4me3 and counteracting heterochromatinization 

in pluripotent cells [81]. However, it was later concluded 

that its association with H3K4me3 is specific to active genes and it 

is in fact exclusively depleted in the dual H3K4/H3K27 regions [82], 

suggesting a selective role as an activator, leaving the suppressed 

bivalent domains intact. Since CHD1 interacts with H3K4me3, it 

raises the possibility that a mechanism to selectively clear CHD1 

from bivalent promoters exists. CHD7 on the other hand was 

shown, using clustering analysis, to be associated with three distinct 

protein complexes in ESCs: an enhancer signature cluster, a c-MYC/ 

n-MYC-enriched cluster, and a PcG cluster, containing SUZ12, 

RING1B, and EZH2 [83], suggesting, among other things, a function 

for CHD7 in PcG-mediated gene regulation. Supporting this notion, 

depletion of the CHD7 homolog Kismet in Drosophila resulted in 

global elevation of H3K27 methylation levels, demonstrating a role 

for CHD7/Kismet in counteracting PcG activity [84]. CHD7 was also 

shown to associate with the PBAF chromatin remodeling complex 

during embryogenesis and human ESC differentiation to promote 

neural crest migration and neural crest gene expression programs 

[85]. CHD7 mutations or other causes of failure to activate neural 

crest migration have been implicated in the development of 

CHARGE syndrome, a complex genetic disease affecting the nervous 

system, heart, vision, ears, and more [86]. These studies highlight a 

link between CHD proteins and H3K27 methylation, thereby 

affecting bivalency albeit indirectly. 

The CHD family of proteins also includes two prominent 

members of the NuRD chromatin remodeling complex, namely 

CHD3 and CHD4. The NuRD complex was shown to be essential for 

proper ESC differentiation [87] and was shown to deacetylate 

H3K27 in ESCs, enabling the subsequent recruitment of PcG 

proteins and trimethylation of H3K27. It therefore controls the 

balance between H3K27 acetylation and methylation, thereby 

enabling cell differentiation [88], although it likely does not act 

directly at bivalent promoters since it is repelled by H3K4me3 [89]. 

Further supporting the tight connection between NuRD and PcG 

during development is the association between CHD4 and the 

H3K27 methyltransferase EZH2 required during astroglial differentiation. 

CHD4 was found to be essential for EZH2 association with 

key astroglial gene promoters, suppressing their expression in nonglial 

cells, and depletion of CHD4 promotes gliogenesis in vivo [90]. 

A similar role for NuRD, mediated by CTBP2, was also seen in differentiating 

ESCs during the exit from pluripotency: NuRD facilitates 

H3K27 deacetylation followed by recruitment of PcG and H3K27 

methylation [91]. Finally, NuRD was also shown to play a role in 

regulating H3K4me3- and H3K27me3-marked bivalent rRNA genes 

[92]. Although the latter study was not performed in ESCs, given 

the importance of the NuRD complex and rRNA transcriptional 

regulation in pluripotency, it is fair to speculate that it likely plays a 

similarly important role there too. Consistent with NuRD’s role in 

regulating bivalency, the complex was shown to interact not only 

with PcG proteins, as discussed above, but also with the H3K4 

methyltransferase MLL1 [93]. But perhaps even more importantly, it 

was also shown to interact with the H3K4 demethylase LSD1, which 

occupies the large majority of active genes, as well as approximately 

two-thirds of bivalent genes (2003 out of 3,094), in ESCs. Thus, 

through its association with the NuRD chromatin remodeling 

complex, LSD1 acts to silence pluripotency genes during early differentiation 

[94]. 

Taken together, these studies demonstrate that chromatin remodeling 

and bivalency, and most significantly PcG-mediated H3K27 

methylation, are oftentimes functionally linked in ESCs and during 

differentiation, acting to resolve bivalent domains into stably activated 

or stably repressed states (Table 1; Fig 4). Because this 

connection between chromatin remodeling and bivalency is only 

beginning to emerge, the examples provided above are sometimes 

sketchy or indirect, with only H3K4 or H3K27 being affected. 

However, as more convincing data are gradually accumulating, it is 

becoming increasingly clear that remodelers, among other chromatin 

factors, act to shape and regulate bivalent chromatin 

Table 1. Chromatin remodelers involved in regulating bivalency. 

Remodeler Complex H3K4me3 H3K27me3 Bivalent References 

Tip60–p400 INO80 U [70] 

BRG1 esBAF U U [71] 

SMARCD1 esBAF U U U [77] 

BAF250a esBAF U U [78] 

CHD7 – U [83] 

CHD3/4 NuRD U U U [88] 

1614 



MLL2 

NuRD 

PRC2 

Sidebar A: 

In need of answers 

? 

esBAF 

H3K4me3 

H3K27me3 

INO80 

(i) Is bivalency a single-cell phenomenon, or a result of population 

heterogeneity? 

(ii) If bivalency is within a single cell, are bivalent histone marks 

present within the same allele? 

(iii) If so, do bivalent histone marks appear within the same nucleosome? 

(iv) If they reside within same nucleosome, are the bivalent marks 

distributed symmetrically or asymmetrically within the histone 

pair of the nucleosome? 

To answer these questions unequivocally, bivalent histone marks must 

be monitored at a single-nucleosome resolution. Single nucleosomes 

can be reconstituted in vitro or digested using micrococcal nuclease 

(MNase) and immobilized for visualization. 

Figure 4. Chromatin remodeling complexes regulating bivalent 

nucleosomes. 

A single schematic bivalent nucleosome is shown (orange) marked with both 

H3K4me3 (green flag, left) and H3K27me3 (red flag, right). Chromatin remodeling 

complexes which were shown to regulate either or both marks are shown in 

green (esBAF), blue (NuRD), and mustard (INO80). Dotted arrows represent 

suggested regulation; dotted double lines represent potential interaction. 

domains. For example, non-canonical, replication-independent 

histone variants including H2A.Z and H3.3 have been recently 

reported to be associated with bivalent chromatin [95–98]. In ESCs, 

H2A.Z is found in active and bivalent promoters, both of which are 

enriched with H3K4me3, but absent from repressed H3K4me3- 

negative promoters [96]. H2A.Z depletion caused derepression of 

poised genes with concomitant loss of PcG components from 

bivalent promoters [95], suggesting a potential co-regulation of 

H2A.Z and PcG. However, a direct interaction between PcG and 

H2A.Z has not been reported so far. Along the same lines, H3.3 was 

shown to be required for the deposition of H3K27 methylation at 

bivalent promoters in a PRC2-dependent manner [98]. When H3.3 

or its chaperone, HIRA, is depleted, H3K27me3 mark is reduced at 

bivalent promoters, along with reduced PRC2 occupancy and 

reduced nucleosome turnover [98], albeit with minimal transcriptional 

changes. This implies that other compensatory mechanisms 

regulating developmental genes in ESCs are in place, but together 

portrays a picture which suggests that histone variants and their 

remodelers might also be a part of the bivalency apparatus. 

Concluding remarks 

Almost a decade has passed since the original discovery of bivalent 

nucleosomes in ESCs [5,6]. While it was tempting to speculate that 

bivalent promoters are restricted to developmentally regulated 

genes, enabling a quick transition to an active or a stable silent 

state, it is clear today that bivalency is more complicated, extending 

to different gene families in multiple different cell types. Furthermore, 

it is increasingly recognized that regulation of the bivalent 

state is highly complex involving a variety of different proteins and 

regulators. Here, we highlighted the family of ATP-dependent chromatin 

remodeling proteins, which is emerging as an important 

player in regulating bivalent domains, especially in the context of 

pluripotent stem cells. While TrxG and PcG proteins provide the 

mechanisms of action for H3K4/H3K27 methylation, we speculate 

(i) Does bivalency play a functional role in ESCs or in any other cell 

type? 

(ii) What is the role of bivalent promoters in terminally differentiated 

cells? 

Complete selective depletion of bivalent domains may be difficult to 

achieve, although a recent study [26] demonstrated little effect on 

early differentiation following depletion of MLL2 which conferred 

selective depletion of H3K4me3 on bivalent promoters. Additional 

studies of selective depletion of bivalent marks through the use of 

genetics or CRISPR/Cas9 approaches will determine the role, if any, 

that bivalent domains play in pluripotency and embryonic development. 

(i) Which of the histone variants are associated with bivalent domains? 

Pull-down of variant modified nucleosomes followed by MS 

approaches, or single nucleosome assays once single-nucleosome resolution 

is achieved, will determine the exact composition/modifications 

of histone variant-containing nucleosomes. 

(i) How do chromatin remodeling proteins regulate bivalent histone 

marks? 

(ii) Do pluripotency factors, in concert with chromatin remodeling 

complexes, regulate bivalent domains? 

Interaction analyses, mutational studies, rescue experiments, and 

functional assays will help achieve mechanistic insights into the regulation 

of bivalent domains by chromatin remodeling proteins and/or 

pluripotency factors. Understanding the mechanism by which chromatin 

remodelers regulate the level and distribution of bivalent chromatin 

domains will be key to establish a direct functional connection 

between chromatin remodeling proteins and bivalency. 

that chromatin remodeling proteins may provide the required bivalent 

specificity. It is important to note that bivalency is not restricted 

to the H3K4me3/H3K27me3 pair; several, albeit more haphazard, 

examples were documented. For example, trophoblast and extraembryonic 

endodermal stem cells were shown to contain a large fraction 

of H3K4me3/H3K9me3 bivalent modifications but little 

H3K4me3/H3K27me3 bivalency [99], and a unique H3K4me1/ 

H3K27ac/H3K9me3 trivalent signature was observed during the 

transition from fibroblasts to induced neurons [100]. In both these 

cases, the “non-canonical” bivalent/trivalent signature enables a 

quick transition from a closed to an open chromatin state akin to 

the situation proposed for the “canonical” K4/K27 marks. The next 

challenge would be to identify meaningful patterns and combinations 

of histone modifications, decipher their potential roles, and 

understand whether such chromatin signatures are the cause or the 

consequence of their suggested function. In addition, programmable 


1615


nucleases (ZFN, TALEN, and especially CRISPR/Cas9, and mutants 

thereof) are already emerging as powerful tools that enable the 

catalysis or removal of specific modifications at bivalent loci, 

making it possible to study downstream effects on the corresponding 

genes [101–104]. More specialized systems such as photoactivatable 

CRISPR switches could take this idea further even to the single 

cell level [105,106]. The combination of epigenetic reprogramming 

assays, single cell technologies, and multilevel epigenomic landscape 

analyses will help decipher the specific roles that multivalent 

domains and their connection with chromatin remodeling play in 

pluripotency, ESCs, and development. 


We thank Bradley Bernstein, Cem Sievers, and Sharon Schlesinger for critical 

comments, and Alva Biran for help with statistics. AH was supported by the 

Nucleosome4D network. We thank the Israel Science Foundation (ISF 1252/12 

and 657/12 to E.M.) and the European Research Council (ERC-281781 to E.M.) 

for financial support. 



References 

1. Kouzarides T (2007) Chromatin modifications and their function. Cell 

128: 693 – 705 

2. Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone 

modifications. Cell Res 21: 381 – 395 

3. Tessarz P, Kouzarides T (2014) Histone core modifications regulating 

nucleosome structure and dynamics. Nat Rev Mol Cell Biol 15: 

703 – 708 

4. Azzaz AM, Vitalini MW, Thomas AS, Price JP, Blacketer MJ, Cryderman 

DE, Zirbel LN, Woodcock CL, Elcock AH, Wallrath LL et al 

(2014) Human heterochromatin protein 1alpha promotes nucleosome 

associations that drive chromatin condensation. J Biol Chem 289: 

6850 – 6861 

5. Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, Gouti M, 

Casanova M, Warnes G, Merkenschlager M et al (2006) Chromatin 

signatures of pluripotent cell lines. Nat Cell Biol 8: 532 – 538 

6. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, 

Meissner A, Wernig M, Plath K et al (2006) A bivalent chromatin structure 

marks key developmental genes in embryonic stem cells. Cell 125: 

315 – 326 

7. Voigt P, Tee WW, Reinberg D (2013) A double take on bivalent promoters. 

Genes Dev 27: 1318 – 1338 

8. Vastenhouw NL, Schier AF (2012) Bivalent histone modifications in 

early embryogenesis. Curr Opin Cell Biol 24: 374 – 386 

9. Dillon N (2012) Factor mediated gene priming in pluripotent stem cells 

sets the stage for lineage specification. BioEssays 34: 194 – 204 

10. Fisher CL, Fisher AG (2011) Chromatin states in pluripotent, differentiated, 

and reprogrammed cells. Curr Opin Genet Dev 21: 140 – 146 

11. Pan G, Tian S, Nie J, Yang C, Ruotti V, Wei H, Jonsdottir GA, Stewart R, 

Thomson JA (2007) Whole-genome analysis of histone H3 lysine 4 and 

lysine 27 methylation in human embryonic stem cells. Cell Stem Cell 1: 

299 – 312 

12. Zhao XD, Han X, Chew JL, Liu J, Chiu KP, Choo A, Orlov YL, Sung WK, 

Shahab A, Kuznetsov VA et al (2007) Whole-genome mapping of 

histone H3 Lys4 and 27 trimethylations reveals distinct genomic 

compartments in human embryonic stem cells. Cell Stem Cell 1: 

286 – 298 

13. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld 

M, Yachechko R, Tchieu J, Jaenisch R et al (2007) Directly reprogrammed 

fibroblasts show global epigenetic remodeling and widespread 

tissue contribution. Cell Stem Cell 1: 55 – 70 

14. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, 

Alvarez P, Brockman W, Kim TK, Koche RP et al (2007) Genome-wide 

maps of chromatin state in pluripotent and lineage-committed cells. 

Nature 448: 553 – 560 

15. Guenther MG, Frampton GM, Soldner F, Hockemeyer D, Mitalipova M, 

Jaenisch R, Young RA (2010) Chromatin structure and gene expression 

programs of human embryonic and induced pluripotent stem cells. Cell 

Stem Cell 7: 249 – 257 

16. Cui K, Zang C, Roh TY, Schones DE, Childs RW, Peng W, Zhao K (2009) 

Chromatin signatures in multipotent human hematopoietic stem cells 

indicate the fate of bivalent genes during differentiation. Cell Stem Cell 

4: 80 – 93 

17. Abraham BJ, Cui K, Tang Q, Zhao K (2013) Dynamic regulation of epigenomic 

landscapes during hematopoiesis. BMC Genom 14: 193 

18. Mohn F, Weber M, Rebhan M, Roloff TC, Richter J, Stadler MB, Bibel M, 

Schubeler D (2008) Lineage-specific polycomb targets and de novo DNA 

methylation define restriction and potential of neuronal progenitors. 

Mol Cell 30: 755 – 766 

19. Roh TY, Cuddapah S, Cui K, Zhao K (2006) The genomic landscape of 

histone modifications in human T cells. Proc Natl Acad Sci USA 103: 

15782 – 15787 

20. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, 

Chepelev I, Zhao K (2007) High-resolution profiling of histone methylations 

in the human genome. Cell 129: 823 – 837 

21. Rodriguez J, Munoz M, Vives L, Frangou CG, Groudine M, Peinado MA 

(2008) Bivalent domains enforce transcriptional memory of DNA 

methylated genes in cancer cells. Proc Natl Acad Sci USA 105: 

19809 – 19814 

22. Bapat SA, Jin V, Berry N, Balch C, Sharma N, Kurrey N, Zhang S, Fang F, 

Lan X, Li M et al (2010) Multivalent epigenetic marks confer microenvironment-responsive 

epigenetic plasticity to ovarian cancer cells. Epigenetics 

5: 716 – 729 

23. McGarvey KM, Van Neste L, Cope L, Ohm JE, Herman JG, Van Criekinge 

W, Schuebel KE, Baylin SB (2008) Defining a chromatin pattern that 

characterizes DNA-hypermethylated genes in colon cancer cells. Cancer 

Res 68: 5753 – 5759 

24. Lin B, Lee H, Yoon JG, Madan A, Wayner E, Tonning S, Hothi P, Schroeder 

B, Ulasov I, Foltz G et al (2015) Global analysis of H3K4me3 and 

H3K27me3 profiles in glioblastoma stem cells and identification of 

SLC17A7 as a bivalent tumor suppressor gene. Oncotarget 6: 5369 – 5381 

25. Denissov S, Hofemeister H, Marks H, Kranz A, Ciotta G, Singh S, 

Anastassiadis K, Stunnenberg HG, Stewart AF (2014) Mll2 is required 

for H3K4 trimethylation on bivalent promoters in embryonic stem cells, 

whereas Mll1 is redundant. Development 141: 526 – 537 

26. Hu D, Garruss AS, Gao X, Morgan MA, Cook M, Smith ER, Shilatifard A 

(2013) The Mll2 branch of the COMPASS family regulates bivalent 

promoters in mouse embryonic stem cells. Nat Struct Mol Biol 20: 

1093 – 1097 

27. Voigt P, LeRoy G, Drury WJ III, Zee BM, Son J, Beck DB, Young NL, 

Garcia BA, Reinberg D (2012) Asymmetrically modified nucleosomes. 

Cell 151: 181 – 193 

1616 



28. Shilatifard A (2012) The COMPASS family of histone H3K4 methylases: 

mechanisms of regulation in development and disease pathogenesis. 

Annu Rev Biochem 81: 65 – 95 

29. Birke M, Schreiner S, Garcia-Cuellar MP, Mahr K, Titgemeyer F, 

Slany RK (2002) The MT domain of the proto-oncoprotein MLL binds to 

CpG-containing DNA and discriminates against methylation. Nucleic 

Acids Res 30: 958 – 965 

30. Bach C, Mueller D, Buhl S, Garcia-Cuellar MP, Slany RK (2009) Alterations 

of the CxxC domain preclude oncogenic activation of mixedlineage 

leukemia 2. Oncogene 28: 815 – 823 

31. Lee JH, Voo KS, Skalnik DG (2001) Identification and characterization of 

the DNA binding domain of CpG-binding protein. J Biol Chem 276: 

44669 – 44676 

32. Clouaire T, Webb S, Skene P, Illingworth R, Kerr A, Andrews R, Lee JH, 

Skalnik D, Bird A (2012) Cfp1 integrates both CpG content and gene 

activity for accurate H3K4me3 deposition in embryonic stem cells. 

Genes Dev 26: 1714 – 1728 

33. Wysocka J, Swigut T, Milne TA, Dou Y, Zhang X, Burlingame AL, Roeder 

RG, Brivanlou AH, Allis CD (2005) WDR5 associates with histone H3 

methylated at K4 and is essential for H3K4 methylation and vertebrate 

development. Cell 121: 859 – 872 

34. Ang YS, Tsai SY, Lee DF, Monk J, Su J, Ratnakumar K, Ding J, Ge Y, 

Darr H, Chang B et al (2011) Wdr5 mediates self-renewal and reprogramming 

via the embryonic stem cell core transcriptional network. 

Cell 145: 183 – 197 

35. Stoller JZ, Huang L, Tan CC, Huang F, Zhou DD, Yang J, Gelb BD, Epstein 

JA (2010) Ash2l interacts with Tbx1 and is required during early 

embryogenesis. Exp Biol Med 235: 569 – 576 

36. Wan M, Liang J, Xiong Y, Shi F, Zhang Y, Lu W, He Q, Yang D, Chen R, 

Liu D et al (2012) The trithorax group protein Ash2l is essential for 

pluripotency and maintaining open chromatin in embryonic stem cells. 

J Biol Chem 288: 5039 – 5048 

37. Biondi CA, Gartside MG, Waring P, Loffler KA, Stark MS, Magnuson MA, 

Kay GF, Hayward NK (2004) Conditional inactivation of the MEN1 gene 

leads to pancreatic and pituitary tumorigenesis but does not affect 

normal development of these tissues. Mol Cell Biol 24: 3125 – 3131 

38. Jiang H, Shukla A, Wang X, Chen WY, Bernstein BE, Roeder RG (2011) 

Role for Dpy-30 in ES cell-fate specification by regulation of H3K4 

methylation within bivalent domains. Cell 144: 513 – 525 

39. Lubitz S, Glaser S, Schaft J, Stewart AF, Anastassiadis K (2007) Increased 

apoptosis and skewed differentiation in mouse embryonic stem cells 

lacking the histone methyltransferase Mll2. Mol Biol Cell 18: 2356 – 2366 

40. Glaser S, Schaft J, Lubitz S, Vintersten K, van der Hoeven F, Tufteland 

KR, Aasland R, Anastassiadis K, Ang SL, Stewart AF (2006) Multiple 

epigenetic maintenance factors implicated by the loss of Mll2 in 

mouse development. Development 133: 1423 – 1432 

41. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones 

RS, Zhang Y (2002) Role of histone H3 lysine 27 methylation in Polycomb-group 

silencing. Science 298: 1039 – 1043 

42. Margueron R, Justin N, Ohno K, Sharpe ML, Son J, Drury WJ III, Voigt P, 

Martin SR, Taylor WR, De Marco V et al (2009) Role of the polycomb 

protein EED in the propagation of repressive histone marks. Nature 

461: 762 – 767 

43. Morey L, Pascual G, Cozzuto L, Roma G, Wutz A, Benitah SA, Di Croce L 

(2012) Nonoverlapping functions of the Polycomb group Cbx family of 

proteins in embryonic stem cells. Cell Stem Cell 10: 47 – 62 

44. O’Loghlen A, Munoz-Cabello AM, Gaspar-Maia A, Wu HA, Banito A, 

Kunowska N, Racek T, Pemberton HN, Beolchi P, Lavial F et al (2012) 

MicroRNA regulation of Cbx7 mediates a switch of Polycomb orthologs 

during ESC differentiation. Cell Stem Cell 10: 33 – 46 

45. Morey L, Aloia L, Cozzuto L, Benitah SA, Di Croce L (2013) RYBP and 

Cbx7 define specific biological functions of polycomb complexes in 

mouse embryonic stem cells. Cell Rep 3: 60 – 69 

46. Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones RS, 

Zhang Y (2004) Role of histone H2A ubiquitination in Polycomb silencing. 

Nature 431: 873 – 878 

47. Cao R, Zhang Y (2004) The functions of E(Z)/EZH2-mediated methylation 

of lysine 27 in histone H3. Curr Opin Genet Dev 14: 155 – 164 

48. Brookes E, de Santiago I, Hebenstreit D, Morris KJ, Carroll T, Xie SQ, 

Stock JK, Heidemann M, Eick D, Nozaki N et al (2012) Polycomb associates 

genome-wide with a specific RNA polymerase II variant, and regulates 

metabolic genes in ESCs. Cell Stem Cell 10: 157 – 170 

49. Min IM, Waterfall JJ, Core LJ, Munroe RJ, Schimenti J, Lis JT (2011) 

Regulating RNA polymerase pausing and transcription elongation in 

embryonic stem cells. Genes Dev 25: 742 – 754 

50. Cooper S, Dienstbier M, Hassan R, Schermelleh L, Sharif J, Blackledge NP, 

De Marco V, Elderkin S, Koseki H, Klose R et al (2014) Targeting polycomb 

to pericentric heterochromatin in embryonic stem cells reveals a 

role for H2AK119u1 in PRC2 recruitment. Cell Rep 7: 1456 – 1470 

51. Ku M, Koche RP, Rheinbay E, Mendenhall EM, Endoh M, Mikkelsen TS, 

Presser A, Nusbaum C, Xie X, Chi AS et al (2008) Genomewide analysis 

of PRC1 and PRC2 occupancy identifies two classes of bivalent 

domains. PLoS Genet 4: e1000242 

52. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, 

Chevalier B, Johnstone SE, Cole MF, Isono K et al (2006) Control of 

developmental regulators by Polycomb in human embryonic stem cells. 

Cell 125: 301 – 313 

53. Thomas LR, Wang Q, Grieb BC, Phan J, Foshage AM, Sun Q, Olejniczak ET, 

Clark T, Dey S, Lorey S et al (2015) Interaction with WDR5 promotes target 

gene recognition and tumorigenesis by MYC. Mol Cell 58: 440 – 452 

54. Ding J, Xu H, Faiola F, Ma’ayan A, Wang J (2011) Oct4 links multiple 

epigenetic pathways to the pluripotency network. Cell Res 22: 155 – 167 

55. Henikoff S, Shilatifard A (2011) Histone modification: cause or cog? 

Trends Genet 27: 389 – 396 

56. Pasini D, Bracken AP, Hansen JB, Capillo M, Helin K (2007) The polycomb 

group protein Suz12 is required for embryonic stem cell differentiation. 

Mol Cell Biol 27: 3769 – 3779 

57. Chamberlain SJ, Yee D, Magnuson T (2008) Polycomb repressive 

complex 2 is dispensable for maintenance of embryonic stem cell 

pluripotency. Stem Cells 26: 1496 – 1505 

58. Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, Yuan GC, Orkin SH 

(2008) EZH1 mediates methylation on histone H3 lysine 27 and 

complements EZH2 in maintaining stem cell identity and executing 

pluripotency. Mol Cell 32: 491 – 502 

59. Leeb M, Pasini D, Novatchkova M, Jaritz M, Helin K, Wutz A (2010) 

Polycomb complexes act redundantly to repress genomic repeats and 

genes. Genes Dev 24: 265 – 276 

60. Faust C, Schumacher A, Holdener B, Magnuson T (1995) The eed mutation 

disrupts anterior mesoderm production in mice. Development 121: 

273 – 285 

61. O’Carroll D, Erhardt S, Pagani M, Barton SC, Surani MA, Jenuwein T 

(2001) The polycomb-group gene Ezh2 is required for early mouse 

development. Mol Cell Biol 21: 4330 – 4336 

62. Pasini D, Bracken AP, Jensen MR, Lazzerini Denchi E, Helin K (2004) 

Suz12 is essential for mouse development and for EZH2 histone 

methyltransferase activity. EMBO J 23: 4061 – 4071 


1617


63. Leeb M, Wutz A (2007) Ring1B is crucial for the regulation of developmental 

control genes and PRC1 proteins but not X inactivation in 

embryonic cells. J Cell Biol 178: 219 – 229 

64. Alkema MJ, van der Lugt NM, Bobeldijk RC, Berns A, van Lohuizen M 

(1995) Transformation of axial skeleton due to overexpression of bmi-1 

in transgenic mice. Nature 374: 724 – 727 

65. van der Lugt NM, Domen J, Linders K, van Roon M, Robanus-Maandag 

E, te Riele H, van der Valk M, Deschamps J, Sofroniew M, van Lohuizen 

M et al (1994) Posterior transformation, neurological abnormalities, 

and severe hematopoietic defects in mice with a targeted deletion of 

the bmi-1 proto-oncogene. Genes Dev 8: 757 – 769 

66. Aloia L, Di Stefano B, Di Croce L (2013) Polycomb complexes in stem 

cells and embryonic development. Development 140: 2525 – 2534 

67. Efroni S, Duttagupta R, Cheng J, Dehghani H, Hoeppner DJ, Dash C, 

Bazett-Jones DP, Le Grice S, McKay RD, Buetow KH et al (2008) Global 

transcription in pluripotent embryonic stem cells. Cell Stem Cell 2: 

437 – 447 

68. Clapier CR, Cairns BR (2009) The biology of chromatin remodeling 

complexes. Annu Rev Biochem 78: 273 – 304 

69. Gaspar-Maia A, Alajem A, Meshorer E, Ramalho-Santos M (2010) Open 

chromatin in pluripotency and reprogramming. Nat Rev Mol Cell Biol 

12: 36 – 47 

70. Fazzio TG, Huff JT, Panning B (2008) An RNAi screen of chromatin 

proteins identifies Tip60-p400 as a regulator of embryonic stem cell 

identity. Cell 134: 162 – 174 

71. Ho L, Jothi R, Ronan JL, Cui K, Zhao K, Crabtree GR (2009) An embryonic 

stem cell chromatin remodeling complex, esBAF, is an essential 

component of the core pluripotency transcriptional network. Proc Natl 

Acad Sci USA 106: 5187 – 5191 

72. Kidder BL, Palmer S, Knott JG (2009) SWI/SNF-Brg1 regulates selfrenewal 

and occupies core pluripotency-related genes in embryonic 

stem cells. Stem Cells 27: 317 – 328 

73. Ho L, Ronan JL, Wu J, Staahl BT, Chen L, Kuo A, Lessard J, Nesvizhskii AI, 

Ranish J, Crabtree GR (2009) An embryonic stem cell chromatin remodeling 

complex, esBAF, is essential for embryonic stem cell self-renewal 

and pluripotency. Proc Natl Acad Sci USA 106: 5181 – 5186 

74. Shen X, Kim W, Fujiwara Y, Simon MD, Liu Y, Mysliwiec MR, Yuan GC, 

Lee Y, Orkin SH (2009) Jumonji modulates polycomb activity and 

self-renewal versus differentiation of stem cells. Cell 139: 1303 – 1314 

75. Landeira D, Sauer S, Poot R, Dvorkina M, Mazzarella L, Jorgensen HF, 

Pereira CF, Leleu M, Piccolo FM, Spivakov M et al (2010) Jarid2 is a 

PRC2 component in embryonic stem cells required for multi-lineage 

differentiation and recruitment of PRC1 and RNA Polymerase II to 

developmental regulators. Nat Cell Biol 12: 618 – 624 

76. Ho L, Miller EL, Ronan JL, Ho WQ, Jothi R, Crabtree GR (2011) esBAF facilitates 

pluripotency by conditioning the genome for LIF/STAT3 signalling 

and by regulating polycomb function. Nat Cell Biol 13: 903 – 913 

77. Alajem A, Biran A, Harikumar A, Sailaja BS, Aaronson Y, Livyatan I, 

Nissim-Rafinia M, Sommer AG, Mostoslavsky G, Gerbasi VR et al (2015) 

Differential association of chromatin proteins identifies BAF60a/ 

SMARCD1 as a regulator of embryonic stem cell differentiation. Cell 

Rep 10: 2019 – 2031 

78. Lei I, West J, Yan Z, Gao X, Fang P, Dennis JH, Gnatovskiy L, Wang W, 

Kingston RE, Wang Z (2015) BAF250a regulates nucleosome occupancy 

and histone modifications in priming embryonic stem cell differentiation. 

J Biol Chem 290: 19343 – 19352 

79. Zhang X, Li B, Li W, Ma L, Zheng D, Li L, Yang W, Chu M, Chen W, 

Mailman RB et al (2014) Transcriptional repression by the BRG1-SWI/ 

SNF complex affects the pluripotency of human embryonic stem cells. 

Stem Cell Rep 3: 460 – 474 

80. Alexander JM, Hota SK, He D, Thomas S, Ho L, Pennacchio LA, Bruneau 

BG (2015) Brg1 modulates enhancer activation in mesoderm lineage 

commitment. Development 142: 1418 – 1430 

81. Gaspar-Maia A, Alajem A, Polesso F, Sridharan R, Mason MJ, 

Heidersbach A, Ramalho-Santos J, McManus MT, Plath K, Meshorer E 

et al (2009) Chd1 regulates open chromatin and pluripotency of 

embryonic stem cells. Nature 460: 863 – 868 

82. Lin JJ, Lehmann LW, Bonora G, Sridharan R, Vashisht AA, Tran N, Plath K, 

Wohlschlegel JA, Carey M (2011) Mediator coordinates PIC assembly 

with recruitment of CHD1. Genes Dev 25: 2198 – 2209 

83. Schnetz MP, Handoko L, Akhtar-Zaidi B, Bartels CF, Pereira CF, Fisher AG, 

Adams DJ, Flicek P, Crawford GE, Laframboise T et al (2010) CHD7 

targets active gene enhancer elements to modulate ES cell-specific 

gene expression. PLoS Genet 6: e1001023 

84. Srinivasan S, Dorighi KM, Tamkun JW (2008) Drosophila Kismet regulates 

histone H3 lysine 27 methylation and early elongation by RNA 

polymerase II. PLoS Genet 4: e1000217 

85. Bajpai R, Chen DA, Rada-Iglesias A, Zhang J, Xiong Y, Helms J, Chang CP, 

Zhao Y, Swigut T, Wysocka J (2010) CHD7 cooperates with PBAF to 

control multipotent neural crest formation. Nature 463: 958 – 962 

86. Hsu P, Ma A, Wilson M, Williams G, Curotta J, Munns CF, Mehr S 

(2014) CHARGE syndrome: a review. J Paediatr Child Health 50: 

504 – 511 

87. Kaji K, Caballero IM, MacLeod R, Nichols J, Wilson VA, Hendrich B 

(2006) The NuRD component Mbd3 is required for pluripotency of 

embryonic stem cells. Nat Cell Biol 8: 285 – 292 

88. Reynolds N, Salmon-Divon M, Dvinge H, Hynes-Allen A, Balasooriya G, 

Leaford D, Behrens A, Bertone P, Hendrich B (2011) NuRD-mediated 

deacetylation of H3K27 facilitates recruitment of Polycomb Repressive 

Complex 2 to direct gene repression. EMBO J 31: 593 – 605 

89. Zegerman P, Canas B, Pappin D, Kouzarides T (2002) Histone H3 lysine 

4 methylation disrupts binding of nucleosome remodeling and 

deacetylase (NuRD) repressor complex. J Biol Chem 277: 11621 – 11624 

90. Sparmann A, Xie Y, Verhoeven E, Vermeulen M, Lancini C, Gargiulo G, 

Hulsman D, Mann M, Knoblich JA, van Lohuizen M (2013) The chromodomain 

helicase Chd4 is required for Polycomb-mediated inhibition 

of astroglial differentiation. EMBO J 32: 1598 – 1612 

91. Kim TW, Kang BH, Jang H, Kwak S, Shin J, Kim H, Lee SE, Lee SM, Lee JH, 

Kim JH et al (2015) Ctbp2 modulates NuRD-mediated deacetylation of 

H3K27 and facilitates PRC2-mediated H3K27me3 in active embryonic 

stem cell genes during exit from pluripotency. Stem Cells 33: 

2442 – 2455 

92. Xie W, Ling T, Zhou Y, Feng W, Zhu Q, Stunnenberg HG, Grummt I, 

Tao W (2012) The chromatin remodeling complex NuRD establishes 

the poised state of rRNA genes characterized by bivalent histone modifications 

and altered nucleosome positions. Proc Natl Acad Sci USA 109: 

8161 – 8166 

93. Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T, Wassell R, 

Dubois G, Mazo A, Croce CM, Canaani E (2002) ALL-1 is a histone 

methyltransferase that assembles a supercomplex of proteins involved 

in transcriptional regulation. Mol Cell 10: 1119 – 1128 

94. Whyte WA, Bilodeau S, Orlando DA, Hoke HA, Frampton GM, Foster CT, 

Cowley SM, Young RA (2012) Enhancer decommissioning by LSD1 

during embryonic stem cell differentiation. Nature 482: 221 – 225 

95. Creyghton MP, Markoulaki S, Levine SS, Hanna J, Lodato MA, Sha K, 

Young RA, Jaenisch R, Boyer LA (2008) H2AZ is enriched at polycomb 

1618 



complex target genes in ES cells and is necessary for lineage commitment. 

Cell 135: 649 – 661 

96. Ku M, Jaffe JD, Koche RP, Rheinbay E, Endoh M, Koseki H, Carr SA, 

Bernstein BE (2012) H2A.Z landscapes and dual modifications in pluripotent 

and multipotent stem cells underlie complex genome regulatory 

functions. Genome Biol 13: R85 

97. Hu G, Cui K, Northrup D, Liu C, Wang C, Tang Q, Ge K, Levens D, 

Crane-Robinson C, Zhao K (2013) H2A.Z facilitates access of active and 

repressive complexes to chromatin in embryonic stem cell self-renewal 

and differentiation. Cell Stem Cell 12: 180 – 192 

98. Banaszynski LA, Wen D, Dewell S, Whitcomb SJ, Lin M, Diaz N, 

Elsasser SJ, Chapgier A, Goldberg AD, Canaani E et al (2013) Hiradependent 

histone H3.3 deposition facilitates PRC2 recruitment at 

developmental loci in ES cells. Cell 155: 107 – 120 

99. Rugg-Gunn PJ, Cox BJ, Ralston A, Rossant J (2010) Distinct histone 

modifications in stem cell lines and tissue lineages from the early 

mouse embryo. Proc Natl Acad Sci USA 107: 10783 – 10790 

100. Wapinski OL, Vierbuchen T, Qu K, Lee QY, Chanda S, Fuentes DR, 

Giresi PG, Ng YH, Marro S, Neff NF et al (2013) Hierarchical mechanisms 

for direct reprogramming of fibroblasts to neurons. Cell 155: 621 – 635 

101. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 

Gonzales AP, Li Z, Peterson RT, Yeh JJ et al (2015) Engineered CRISPR- 

Cas9 nucleases with altered PAM specificities. Nature 523: 481 – 485 

102. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim 

WA (2013) Repurposing CRISPR as an RNA-guided platform for 

sequence-specific control of gene expression. Cell 152: 1173 – 1183 

103. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan 

S, Shivalila CS, Dadon DB, Jaenisch R (2013) Multiplexed activation of 

endogenous genes by CRISPR-on, an RNA-guided transcriptional activator 

system. Cell Res 23: 1163 – 1171 

104. Hu J, Lei Y, Wong WK, Liu S, Lee KC, He X, You W, Zhou R, Guo JT, 

Chen X et al (2014) Direct activation of human and mouse Oct4 genes 

using engineered TALE and Cas9 transcription factors. Nucleic Acids Res 

42: 4375 – 4390 

105. Nihongaki Y, Kawano F, Nakajima T, Sato M (2015) Photoactivatable 

CRISPR-Cas9 for optogenetic genome editing. Nat Biotechnol 33: 

755 – 760 

106. Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M (2015) CRISPR- 

Cas9-based photoactivatable transcription system. Chem Biol 22: 

169 – 174 


1619


TRANSPARENT 

PROCESS 

OPEN 

ACCESS 

CRISPR/Cas9‐induced disruption of gene expression 

in mouse embryonic brain and single neural stem 

cells in vivo 

Nereo Kalebic 1 , † , Elena Taverna 1 , † , Stefania Tavano 1 , Fong Kuan Wong 1 , Dana Suchold 1 , Sylke Winkler 1 , 

Wieland B Huttner*, 1 and Mihail Sarov*, 1 

1 

Max Planck Institute of Molecular Cell Biology and Genetics (MPI‐CBG), Dresden, Germany 

* 

Corresponding author. Tel: +49 3512101500; E‐mail: huttner@mpi-cbg.de 

Corresponding author. Tel: +49 3512102617; E‐mail: sarov@mpi-cbg.de 

† 

These authors contributed equally to this work 

DOI 10.15252/embr.201541715 | Published online 12.01.2016 

EMBO reports (2016) 17, 338-348 

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing 

mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide 

RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock‐in mice, expression of GFP, which occurs specifically 

in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny 

of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, 

near‐maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using 

microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained 

disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation 

or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical 

neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the 

present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring 

disruption of expression of specific genes to dissect their role in mammalian brain development. 

Synopsis 

This study shows that the CRISPR/Cas9 system can be used to disrupt gene expression 

in single neural stem cells and their progeny in the embryonic mammalian brain in vivo. 

This can be achieved by electroporation of a single plasmid or of protein complexes. 

•• 

In utero electroporation of a single plasmid encoding Cas9 and a gRNA into the brain of 

mouse embryos leads to gene inactivation in the immediate progeny of the targeted neural 

stem cells. 

•• 

Disruption of gene expression can also be achieved by electroporating the Cas9 protein in a 

complex with gRNA directly into embryonic mouse brains in vivo. 

•• 

Microinjection of a complex of Cas9 protein and gRNA into single neural stem cells in 

organotypic slice culture results in disruption of gene expression within one cell cycle.

Article 

SOURCE 

DATA 

OPEN 

ACCESS 

Integrative genomics positions MKRN1 as a novel 

ribonucleoprotein within the embryonic stem cell 

gene regulatory network 

Paul A Cassar 1 , 2 , † , Richard L Carpenedo 3 , † , Payman Samavarchi‐Tehrani 4 , Jonathan B Olsen 2 , 4 , Chang Jun 

Park 5 , Wing Y Chang 3 , Zhaoyi Chen 3 , 6 , Chandarong Choey 3 , Sean Delaney 3 , 6 , Huishan Guo 7 , Hongbo Guo 8 , R 

Matthew Tanner 3 , 6 , Theodore J Perkins 3 , 7 , Scott A Tenenbaum 9 , Andrew Emili 2 , 4 , 8 , Jeffrey L Wrana 2 , 4 , 10 , Derrick 

Gibbings 7 and William L Stanford*, 1 , 2 , 3 , 5 , 6 , 7 , 11 

1 

Institute of Medical Science University of Toronto, Toronto, ON, Canada 2 Collaborative Program in Genome Biology and Bioinformatics, University of Toronto, 

Toronto, ON, Canada 3 Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, Canada 4 Department of Molecular Genetics, 

University of Toronto, Toronto, ON, Canada 5 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada 6 Department of 

Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada 7 Department of Biochemistry, Microbiology & Immunology, University of Ottawa, 

Ottawa, ON, Canada 8 Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, ON, Canada 9 Colleges of Nanoscale 

Science & Engineering SUNY Polytechnic Institute, Albany, NY, USA 10 Center for Systems Biology, Lunenfeld‐Tanenbaum Research Institute, Mount Sinai Hospital, 

Toronto, ON, Canada 11 Ottawa Institute of Systems Biology, Ottawa, ON, Canada 

* 

Corresponding author. Tel: +1 613 737 8899 Ext. 75495; E‐mail: wstanford@ohri.ca 

† 

These authors contributed equally to this study 


EMBO reports (2015) 16, 1334-1357 

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC 

identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous 

temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin‐1 (MKRN1) 

as a potential novel component of the ESC GRN. Here, using multilayered systems‐level analyses, we compiled a 

MKRN1‐centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in 

undifferentiated ESCs revealed that MKRN1 associates with RNA‐binding proteins, and ensuing RIP‐chip analysis 

determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that 

function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule‐resident 

protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our 

unbiased systems‐level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN. 

Synopsis 

An unbiased integrative genomics approach identified proteins associated with the E3 

ubiquitin ligase MKRN1 and its associated mRNAs in ESCs, indicating that MKRN1 is a 

RNA‐binding protein involved in the modulation of cellular stress and apoptosis. 

•• 

MKRN1 is present in ribonucleoprotein particles in ESCs along with other RNA‐binding 

proteins that are also found in stress granules, including PABPC1, PABPC4 and YBX1. 

•• 

MKRN1 associates with mRNAs encoding proteins that function during cellular stress and 

apoptosis. 

•• 

Loss of MKRN1 augments apoptosis in ESCs recovering from stress. 

•• 

However, MKRN1 is not required for survival in unstressed ESCs.

Article 

TRANSPARENT 

PROCESS 

SOURCE 

DATA 

OPEN 

ACCESS 

Selective influence of Sox2 on POU transcription 

factor binding in embryonic and neural stem cells 

Tapan Kumar Mistri 1 , 2 , 3 , Arun George Devasia 2 , Lee Thean Chu 2 , Wei Ping Ng 1 , Florian Halbritter 3 , Douglas 

Colby 3 , Ben Martynoga 4 , Simon R Tomlinson 3 , Ian Chambers*, 3 , Paul Robson*, 2 , 5 , 6 and Thorsten Wohland*, 1 , 5 , 7 

1 

Department of Chemistry, National University of Singapore, Singapore, Singapore 

2 

Developmental Cellomics Laboratory, Genome Institute of Singapore, Singapore, Singapore 

3 

MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK 

4 

Division of Molecular Neurobiology, MRC‐National Institute for Medical Research, Mill Hill, London, UK 

5 

Department of Biological Sciences, National University of Singapore, Singapore, Singapore 

6 

The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA 

7 

Centre for Bioimaging Sciences, National University of Singapore, Singapore, Singapore 

* 

Corresponding author. Tel: +44 131 651 9500; E‐mail: ichambers@ed.ac.uk 

Corresponding author. Tel: +1 207 288 6594; E‐mail: paul.robson@jax.org 

Corresponding author. Tel: +65 6516 1248; Fax: +65 6776 7882; E‐mail: twohland@nus.edu.sg 


EMBO reports (2015) 16, 1177-1191 

Embryonic stem cell (ESC) identity is orchestrated by co‐operativity between the transcription factors (TFs) Sox2 and 

the class V POU‐TF Oct4 at composite Sox/Oct motifs. Neural stem cells (NSCs) lack Oct4 but express Sox2 and class 

III POU‐TFs Oct6, Brn1 and Brn2. This raises the question of how Sox2 interacts with POU‐TFs to transcriptionally 

specify ESCs versus NSCs. Here, we show that Oct4 alone binds the Sox/Oct motif and the octamer‐containing 

palindromic MORE equally well. Sox2 binding selectively increases the affinity of Oct4 for the Sox/Oct motif. In 

contrast, Oct6 binds preferentially to MORE and is unaffected by Sox2. ChIP‐Seq in NSCs shows the MORE to be the 

most enriched motif for class III POU‐TFs, including MORE subtypes, and that the Sox/Oct motif is not enriched. 

These results suggest that in NSCs, co‐operativity between Sox2 and class III POU‐TFs may not occur and that 

POU‐TF‐driven transcription uses predominantly the MORE cis architecture. Thus, distinct interactions between 

Sox2 and POU‐TF subclasses distinguish pluripotent ESCs from multipotent NSCs, providing molecular insight into 

how Oct4 alone can convert NSCs to pluripotency. 

Synopsis 

Sox2 and POU‐TF subclasses have distinct interactions that distinguish pluripotent ESC 

from multipotent NSC. The different combinations of TFs and DNA binding motifs in ESC 

and NSC might explain why Oct4 alone can convert NSC to pluripotency. 

•• 

Oct4 binds the Sox/Oct motif and the palindromic MORE equally well, whereas Oct6 binds 

preferentially to MORE. 

•• 

Sox2 binding selectively increases the affinity of Oct4 for the Sox/Oct motif but shows no 

effect on Oct6. 

•• 

The MORE is the most enriched motif for class III POU‐TFs Oct6, Brn1 and Brn2 in NSC 

whereas the Sox/Oct motif is the most enriched motif for both Oct4 and Sox2 in ESC.

Article 

TRANSPARENT 

PROCESS 

Mitochondrial metabolism in hematopoietic stem 

cells requires functional FOXO3 

Pauline Rimmelé 1 , † , Raymond Liang 1 , 2 , † , Carolina L Bigarella 1 , † , Fatih Kocabas 3 , Jingjing Xie 3 , Madhavika N 

Serasinghe 4 , Jerry Chipuk 4 , 5 , Hesham Sadek 3 , 6 , Cheng Cheng Zhang 6 and Saghi Ghaffari*, 1 , 2 , 5 , 7 , 8 

1 

Department of Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 

2 

Developmental and Stem Cell Biology Multidisciplinary Training Area, Icahn School of Medicine at Mount Sinai, New York, NY, USA 

3 

Division of Cardiology, UT Southwestern Medical Center, Dallas, TX, USA 

4 

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA 

5 

Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 

6 

Department of Physiology, UT Southwestern Medical Center, Dallas, TX, USA 

7 

Division of Hematology, Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA 

8 

Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 

* 

Corresponding author. Tel: +1 212 659 8271; Fax: +1 212 803 6740; E‐mail: saghi.ghaffari@mssm.edu 

† 

These authors contributed equally to this work 


EMBO reports (2015) 16, 1164-1176 

Hematopoietic stem cells (HSC) are primarily dormant but have the potential to become highly active on 

demand to reconstitute blood. This requires a swift metabolic switch from glycolysis to mitochondrial oxidative 

phosphorylation. Maintenance of low levels of reactive oxygen species (ROS), a by‐product of mitochondrial 

metabolism, is also necessary for sustaining HSC dormancy. Little is known about mechanisms that integrate 

energy metabolism with hematopoietic stem cell homeostasis. Here, we identify the transcription factor FOXO3 as a 

new regulator of metabolic adaptation of HSC. ROS are elevated in Foxo3 –/– HSC that are defective in their activity. 

We show that Foxo3 –/– HSC are impaired in mitochondrial metabolism independent of ROS levels. These defects are 

associated with altered expression of mitochondrial/metabolic genes in Foxo3 –/– hematopoietic stem and progenitor 

cells (HSPC). We further show that defects of Foxo3 –/– HSC long‐term repopulation activity are independent of ROS 

or mTOR signaling. Our results point to FOXO3 as a potential node that couples mitochondrial metabolism with HSC 

homeostasis. These findings have critical implications for mechanisms that promote malignant transformation and 

aging of blood stem and progenitor cells. 

Synopsis 

FOXO3 regulates oxidative stress in LT‐HSC, which are highly sensitive to increased reactive 

oxygen species. However, while the impaired function of Foxo3 –/– LT‐HSC is associated 

with defective mitochondrial metabolism, it is not mediated by oxidative stress or mTOR 

signaling. 

•• 

Mitochondrial metabolism is impaired in Foxo3 –/– LT‐HSCs. 

•• 

Defects in Foxo3 –/– LT‐HSC activity in vivo or Foxo3 –/– LT‐HSC mitochondrial function are not 

mediated by oxidative stress.


TRANSPARENT 

PROCESS 

DAZL regulates Tet1 translation in murine 

embryonic stem cells 

Maaike Welling 1 , † , Hsu‐Hsin Chen 2 , 3 , † , Javier Muñoz 4 , 511 , Michael U Musheev 6 , Lennart Kester 1 , Jan Philipp 

Junker 1 , Nikolai Mischerikow 4 , 512 , Mandana Arbab 1 , Ewart Kuijk 1 , Lev Silberstein 2 , 3 , Peter V Kharchenko 7 , 

Mieke Geens 8 , Christof Niehrs 6 , 9 , Hilde van de Velde 8 , Alexander van Oudenaarden 1 , Albert JR Heck 4 , 5 and 

Niels Geijsen*, 1 , 10 

1 

Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, The Netherlands 

2 

Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA 

3 

Harvard Stem Cell Institute, Cambridge, MA, USA 

4 

Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, 

Utrecht University, Utrecht, The Netherlands 

5 

Netherlands Proteomics Centre, Utrecht, The Netherlands 

6 

Institute of Molecular Biology, Mainz, Germany 

7 

Center for Biomedical Informatics, Harvard Medical School, Boston, MA, USA 

8 

Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Brussels, Belgium 

9 

Division of Molecular Embryology, DKFZ‐ZMBH Alliance, Heidelberg, Germany 

10 

Department of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 

11 

Proteomics Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 

12 

Research Institute of Molecular Pathology, Mass Spectrometry & Protein Chemistry, Vienna, Austria 

* 

Corresponding author. Tel: +31 30 2121800; E‐mail: n.geijsen@hubrecht.eu 

† 

These authors contributed equally to this manuscript 


EMBO reports (2015) 16, 791-802 

Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve 

pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so‐called 2i conditions) 

pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this 

naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA‐binding protein known to 

play a key role in germ‐cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve 

pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We 

demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5‐hydroxylation of methyl‐cytosine, and enhances 

Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein 

levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i‐mediated 

TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve 

pluripotency and demonstrate that DAZL enhances TET1‐mediated cytosine hydroxymethylation in ESCs that are 

actively reprogramming to a pluripotent ground state. 

Synopsis 

This study reports that by enhancing Tet1 mRNA translation, the RNA‐binding protein 

DAZL regulates TET1‐mediated cytosine hydroxymethylation in ESCs during active 

reprogramming to a pluripotent ground state. 

•• 

DAZL marks ESCs actively transitioning to a naïve pluripotent state. 

•• 

DAZL enhances Tet1 mRNA translation. 

•• 

DAZL overexpression results in increased hydroxymethylation.


TRANSPARENT 

PROCESS 

Distinct germline progenitor subsets defined 

through Tsc2–mTORC1 signaling 

Robin M Hobbs*, 1 , 2 , Hue M La 2 , Juho‐Antti Mäkelä 2 , Toshiyuki Kobayashi 3 , Tetsuo Noda 4 and 

Pier Paolo Pandolfi*, 1 

1 

Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center Harvard Medical 

School, Boston, MA, USA 

2 

Australian Regenerative Medicine Institute and Department of Anatomy and Developmental Biology Monash University, Clayton, VIC, Australia 

3 

Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo, Japan 

4 

Department of Cell Biology, JFCR Cancer Institute, Tokyo, Japan 

* 

Corresponding author. Tel: +1 617 735 2145; Fax: +1 617 735 2120; E‐mail: ppandolf@bidmc.harvard.edu 

Corresponding author. Tel: +61 3 9902 9611; Fax: +61 3 9902 9729; E‐mail: robin.hobbs@monash.edu 


EMBO reports (2015) 16, 467-480 

Adult tissue maintenance is often dependent on resident stem cells; however, the phenotypic and functional 

heterogeneity existing within this self‐renewing population is poorly understood. Here, we define distinct subsets of 

undifferentiated spermatogonia (spermatogonial progenitor cells; SPCs) by differential response to hyperactivation 

of mTORC1, a key growth‐promoting pathway. We find that conditional deletion of the mTORC1 inhibitor Tsc2 

throughout the SPC pool using Vasa‐Cre promotes differentiation at the expense of self‐renewal and leads to 

germline degeneration. Surprisingly, Tsc2 ablation within a subset of SPCs using Stra8‐Cre did not compromise 

SPC function. SPC activity also appeared unaffected by Amh‐Cre‐mediated Tsc2 deletion within somatic cells of 

the niche. Importantly, we find that differentiation‐prone SPCs have elevated mTORC1 activity when compared to 

SPCs with high self‐renewal potential. Moreover, SPCs insensitive to Tsc2 deletion are preferentially associated with 

mTORC1‐active committed progenitor fractions. We therefore delineate SPC subsets based on differential mTORC1 

activity and correlated sensitivity to Tsc2 deletion. We propose that mTORC1 is a key regulator of SPC fate and 

defines phenotypically distinct SPC subpopulations with varying propensities for self‐renewal and differentiation. 

Synopsis 

This study defines a cell‐autonomous role for mTORC1 signaling in balanced self‐renewal 

and differentiation of male germline progenitors (SPCs). Within the SPC pool, mTORC1 

activation is preferentially observed within differentiation‐prone fractions and promotes 

differentiation at the expense of self‐renewal. 

•• 

SPCs exhibit heterogeneous activation of mTORC1 in vivo; cells with high self‐renewal 

potential have low pathway activity, while differentiation‐prone subsets have higher activity. 

•• 

mTORC1 activation driven by conditional Tsc2 deletion triggers differentiation commitment 

and depletes the progenitor pool. 

•• 

mTORC1‐active committed progenitors display increased activity of the germ cell‐specific 

Stra8‐Cre transgene and are insensitive to Tsc2 deletion and further mTORC1 activation.


TRANSPARENT 

PROCESS 

Epigenetic predisposition to reprogramming fates in 

somatic cells 

Maayan Pour 1 , † , Inbar Pilzer 1 , † , Roni Rosner 1 , Zachary D Smith 2 , Alexander Meissner 2 and Iftach Nachman*, 1 

1 

Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv, Israel 

2 

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA 

* 

Corresponding author. Tel: +972 3 640 5900; E‐mail: iftachn@post.tau.ac.il 



EMBO reports (2015) 16, 370-378 

Reprogramming to pluripotency is a low‐efficiency process at the population level. Despite notable advances to 

molecularly characterize key steps, several fundamental aspects remain poorly understood, including when the 

potential to reprogram is first established. Here, we apply live‐cell imaging combined with a novel statistical approach 

to infer when somatic cells become fated to generate downstream pluripotent progeny. By tracing cell lineages 

from several divisions before factor induction through to pluripotent colony formation, we find that pre‐induction 

sister cells acquire similar outcomes. Namely, if one daughter cell contributes to a lineage that generates induced 

pluripotent stem cells (iPSCs), its paired sibling will as well. This result suggests that the potential to reprogram 

is predetermined within a select subpopulation of cells and heritable, at least over the short term. We also find 

that expanding cells over several divisions prior to factor induction does not increase the per‐lineage likelihood 

of successful reprogramming, nor is reprogramming fate correlated to neighboring cell identity or cell‐specific 

reprogramming factor levels. By perturbing the epigenetic state of somatic populations with Ezh2 inhibitors prior 

to factor induction, we successfully modulate the fraction of iPSC‐forming lineages. Our results therefore suggest 

that reprogramming potential may in part reflect preexisting epigenetic heterogeneity that can be tuned to alter the 

cellular response to factor induction. 

Synopsis 

The potential of mouse embryonic fibroblasts (MEFs) to generate iPSCs is determined 

before OKSM induction and symmetrically maintained over the short term. Epigenetic 

perturbations of MEFs can alter their future response to reprogramming. 

•• 

The potential to generate iPSC colonies is shared between sister lineages emanating from a 

pre‐induction cell division. 

•• 

Cell‐specific OKSM levels or local niche effects do not explain preference toward iPSC fate. 

•• 

Perturbing H3K27 or H3K4 methylation marks prior to OKSM induction increases the 

number of iPSC lineages.

Review 

Harnessing the apoptotic programs in cancer 

stem‐like cells 

Ying‐Hua Wang 1 , 2 , 3 and David T Scadden*, 1 , 2 , 3 

1 

Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA, USA 

2 

Harvard Stem Cell Institute, Cambridge, MA, USA 

3 

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA 

* 

Corresponding author. Tel: +1 617 7265615; E‐mail: dscadden@mgh.harvard.edu 


EMBO reports (2015) 16, 1084-1098 

Elimination of malignant cells is an unmet challenge for most human cancer types even with therapies targeting 

specific driver mutations. Therefore, a multi‐pronged strategy to alter cancer cell biology on multiple levels is 

increasingly recognized as essential for cancer cure. One such aspect of cancer cell biology is the relative apoptosis 

resistance of tumor‐initiating cells. Here, we provide an overview of the mechanisms affecting the apoptotic 

process in tumor cells emphasizing the differences in the tumor‐initiating or stem‐like cells of cancer. Further, we 

summarize efforts to exploit these differences to design therapies targeting that important cancer cell population. 

Synopsis 

Cancer stem-like cells are less sensitive to apoptotic signals and 

more resistant to therapy. This review discusses the regulation of 

apoptosis in CSCs, focusing on the therapeutical modulation of 

pro- and anti-apoptotic pathways to induce CSC death.

Review 

Effects of inflammation on stem cells: 

together they strive? 

Caghan Kizil*,1,2,†, Nikos Kyritsis2,† and Michael Brand*,2 

1 

German Centre for Neurodegenerative Diseases (DZNE) Dresden within the Helmholtz Association, Dresden, Germany 

2 

DFG‐Center for Regenerative Therapies Dresden, Cluster of Excellence (CRTD) of the Technische Universität Dresden, Dresden, Germany 

* Corresponding author. Tel: +49 351 458 82315; E‐mail: caghan.kizil@dzne.de 

Corresponding author. Tel: +49 351 458 82300; E‐mail: michael.brand@biotec.tu-dresden.de 



EMBO reports (2015) 16, 416-426 

Inflammation entails a complex set of defense mechanisms acting in concert to restore the homeostatic balance in 

organisms after damage or pathogen invasion. This immune response consists of the activity of various immune 

cells in a highly complex manner. Inflammation is a double‐edged sword as it is reported to have both detrimental 

and beneficial consequences. In this review, we discuss the effects of inflammation on stem cell activity, focusing 

primarily on neural stem/progenitor cells in mammals and zebrafish. We also give a brief overview of the effects 

of inflammation on other stem cell compartments, exemplifying the positive and negative role of inflammation 

on stemness. The majority of the chronic diseases involve an unremitting phase of inflammation due to improper 

resolution of the initial pro‐inflammatory response that impinges on the stem cell behavior. Thus, understanding 

the mechanisms of crosstalk between the inflammatory milieu and tissue‐resident stem cells is an important basis 

for clinical efforts. Not only is it important to understand the effect of inflammation on stem cell activity for further 

defining the etiology of the diseases, but also better mechanistic understanding is essential to design regenerative 

therapies that aim at micromanipulating the inflammatory milieu to offset the negative effects and maximize the 

beneficial outcomes. 

Synopsis 

Inflammation is an important defense mechanism against 

pathogens or tissue damage, but it can also have detrimental 

consequences. This review focuses specifically on the effects of 

inflammation on stem cells.

NOTES

FURTHER further reading READING 

The EMBO Journal 

Articles 

Stat3 promotes mitochondrial 

transcription and oxidative 

respiration during maintenance 

and induction of naive 

pluripotency 

Elena Carbognin, Riccardo M Betto, 

Maria E Soriano, Austin G Smith, 

Graziano Martello 

DOI 10.15252/embj.201592629 

Published online: 22.02.2016 

Oct4-induced oligodendrocyte 

progenitor cells enhance 

functional recovery in spinal 

cord injury model 

Jeong Beom Kim, Hyunah Lee, Marcos J 

Araúzo-Bravo, Kyujin Hwang, Donggyu 

Nam, Myung Rae Park, Holm Zaehres, 

Kook In Park, Seok-Jin Lee 

DOI 10.15252/embj.201592652 


Notch signaling regulates 

gastric antral LGR5 stem cell 

function 

Elise S Demitrack, Gail B Gifford, 

Theresa M Keeley, Alexis J Carulli, Kelli 

L VanDussen, Dafydd Thomas, Thomas 

J Giordano, Zhenyi Liu, Raphael Kopan, 

Linda C Samuelson 

DOI:10.15252/embj.201490583 


The tRNA methyltransferase 

Dnmt2 is required for accurate 

polypeptide synthesis during 

haematopoiesis 

Francesca Tuorto, Friederike Herbst, 

Nader Alerasool, Sebastian Bender, 

Oliver Popp, Giuseppina Federico, 

Sonja Reitter, Reinhard Liebers, Georg 

Stoecklin, Hermann-Josef Gröne, 

Gunnar Dittmar, Hanno Glimm, Frank 

Lyko 

DOI:10.15252/embj.201591382 


Robust intestinal homeostasis 

relies on cellular plasticity in 

enteroblasts mediated by miR- 

8–Escargot switch 

Zeus A Antonello, Tobias Reiff, Esther 

Ballesta-Illan, Maria Dominguez 

DOI:10.15252/embj.201591517 


Neuropeptide Y regulates 

the hematopoietic stem cell 

microenvironment and prevents 

nerve injury in the bone marrow 

Min Hee Park, Hee Kyung Jin, Woo-Kie 

Min, Won Woo Lee, Jeong Eun Lee, 

Haruhiko Akiyama, Herbert Herzog, 

Grigori N Enikolopov, Edward H 

Schuchman, Jae-sung Bae 

DOI:10.15252/embj.201490174 


Controlled induction of human 

pancreatic progenitors produces 

functional beta-like cells in vitro 

Holger A Russ, Audrey V Parent, Jennifer 

J Ringler, Thomas G Hennings, Gopika 

G Nair, Mayya Shveygert, Tingxia Guo, 

Sapna Puri, Leena Haataja, Vincenzo 

Cirulli, Robert Blelloch, Greg L Szot, 

Peter Arvan, Matthias Hebrok 

DOI:10.15252/embj.201591058 


Sox2, Tlx, Gli3, and Her9 

converge on Rx2 to define retinal 

stem cells in vivo 

Robert Reinhardt, Lázaro Centanin, 

Tinatini Tavhelidse, Daigo Inoue, Beate 

Wittbrodt, Jean-Paul Concordet, Juan 

Ramón Martinez-Morales, Joachim 

Wittbrodt 

DOI:10.15252/embj.201490706 


Telomerase abrogates 

aneuploidy-induced telomere 

replication stress, senescence 

and cell depletion 

Meena, Jitendra K.; Cerutti, Aurora; 

Beichler, Christine; Morita, Yohei; 

Bruhn, Christopher; Kumar, Mukesh; 

Kraus, Johann M.; Speicher, Michael 

R.; Wang, Zhao-Qi; Kestler, Hans A.; 

di Fagagna, Fabrizio d’Adda; Guenes, 

Cagatay; Rudolph, Karl Lenhard 

DOI: 10.15252/embj.201490070 


Smg6/Est1 licenses embryonic 

stem cell differentiation via 

nonsense–mediated mRNA 

decay 

Tangliang Li, Yue Shi, Pei Wang, Luis 

Miguel Guachalla, Baofa Sun, Tjard 

Joerss, Yu-Sheng Chen, Marco Groth, 

Anja Krueger, Matthias Platzer, Yun-Gui 

Yang, Karl Lenhard Rudolph, Zhao-Qi 

Wang 

DOI:10.15252/embj.201489947 


Snai1 regulates cell lineage 

allocation and stem cell 

maintenance in the mouse 

intestinal epithelium 

Katja Horvay, Thierry Jardé, Franca 

Casagranda, Victoria M Perreau, 

Katharina Haigh, Christian M Nefzger, 

Reyhan Akhtar, Thomas Gridley, Geert 

Berx, Jody J Haigh, Nick Barker, Jose M 

Polo, Gary R Hime, Helen E Abud 

DOI 10.15252/embj.201490881 


Human primordial germ cell 

commitment in vitro associates 

with a unique PRDM14 

expression profile 

Fumihiro Sugawa, Marcos J Araúzo- 

Bravo, Juyong Yoon, Kee-Pyo Kim, 

Shinya Aramaki, Guangming Wu, 

Martin Stehling, Olympia E Psathaki, 

Karin Hübner, Hans R Schöler 

DOI 10.15252/embj.201488049 


emboj.embopress.org 

EMBO Molecular Medicine 

Research Articles 

Aberrant epigenome in iPSCderived 

dopaminergic neurons 

from Parkinson’s disease 

patients 

Rubén Fernández-Santiago, Iria 

Carballo-Carbajal, Giancarlo Castellano, 

Roger Torrent, Yvonne Richaud, Adriana 

Sánchez-Danés, Roser Vilarrasa-Blasi, 

Alex Sánchez-Pla, José Luis Mosquera, 

Jordi Soriano, José López-Barneo, Josep 

M Canals, Jordi Alberch, Ángel Raya, 

Miquel Vila, Antonella Consiglio, José 

I Martín-Subero, Mario Ezquerra, 

Eduardo Tolosa 

DOI 10.15252/emmm.201505439 | 


The MICA-129 dimorphism 

affects NKG2D signaling and 

outcome of hematopoietic stem 

cell transplantation 

Antje Isernhagen, Dörthe Malzahn, 

Elena Viktorova, Leslie Elsner, Sebastian 

Monecke, Frederike von Bonin, Markus 

Kilisch, Janne Marieke Wermuth, Neele 

Walther, Yesilda Balavarca, Christiane 

Stahl-Hennig, Michael Engelke, Lutz 

Walter, Heike Bickeböller, Dieter Kube, 

Gerald Wulf, Ralf Dressel 

DOI 10.15252/emmm.201505246 | 


Reviews 

Reprogramming and 

transdifferentiation for 

cardiovascular development and 

regenerative medicine: where 

do we stand? 

Antje D Ebert, Sebastian Diecke, Ian Y 

Chen, Joseph C Wu 

DOI 10.15252/emmm.201504395 | 


Lost in translation: pluripotent 

stem cell-derived hematopoiesis 

Mania Ackermann, Steffi Liebhaber, 

Jan-Henning Klusmann, Nico Lachmann 

DOI 10.15252/emmm.201505301 | 


embomolmed.embopress.org 

Molecular Systems Biology 

Articles 

The bimodally expressed 

microRNA miR-142 gates exit 

from pluripotency 

Hanna L Sladitschek, Pierre A Neveu 

DOI 10.15252/msb.20156525 | 


Hierarchical folding and 

reorganization of chromosomes 

are linked to transcriptional 

changes in cellular 

differentiation 

James Fraser, Carmelo Ferrai, Andrea 

M Chiariello, Markus Schueler, Tiago 

Rito, Giovanni Laudanno, Mariano 

Barbieri, Benjamin L Moore, Dorothee 

CA Kraemer, Stuart Aitken, Sheila Q 

Xie, KellyJ Morris, Masayoshi Itoh, 

Hideya Kawaji, InesJaeger, Yoshihide 

Hayashizaki, Piero Carninci, Alistair RR 

Forrest, , Colin A Semple, Josée Dostie, 

Ana Pombo, Mario Nicodemi 

DOI 10.15252/msb.20156492 | 


Review 

Cell dynamics and gene 

expression control in tissue 

homeostasis and development 

Pau Rué, Alfonso Martinez Arias 

DOI 10.15252/msb.20145549 | 


msb.embopress.org

embor.embopress.org 

217407 / MLEA033411

Stem Cells

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?