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R. Negri - Role of the presumptive human transcription factor Gas41
sitivity to low concentration of cesium of the yaf9∆
strain was not observed in the hog1∆-yaf9∆ double
mutant. Finally, we showed that the osmotic activity
of cesium salt was detected and signaled by the two
branches downstream of the Sln1 and Sho1 sensors
of the HOG pathway, but the cesium-specific
response mediated by Yaf9, that counteracted the
efficiency of the HOG pathway, was not routed by
these sensors. In conclusion, the cesium specific
response of S. cerevisiae involved the Yaf9 protein
and its activity of chromatin remodelling and transcription
regulation.
Genome-wide localization of Yaf9
in S. cerevisiae
We constructed a yeast strain carrying a C-terminal
HA tag fused to Yaf9: the fusion protein could suppress
the phenotypes of the yaf9∆ strain. We performed
a genome-wide localization of Yaf9 by ChIP
on chip analysis, using antibodies against the HA tag
and control experiments on the wild type strain. The
immunoprecipitated DNA was used to hybridize
microarray slides containing the whole S. cerevisiae
genome. Data were normalized and analysed with
microarray-dedicated data mining softwares. The
main conclusions of this analysis are the following:
1) the genome-wide localization of Yaf9 is similar to
that one of the NuA4 histone acetylation complex; 2)
the localization is not limited to promoter regions
but extends to ORFs; 3) there is a positive and significant
correlation between ORF occupancy and
transcription level; 4) Yaf9 binds significantly to centromeric
regions.
It should be noted that the experimental background
was high. In fact conclusions 3 and 4 seem to be partially
applicable also to the tag-less wild type control.
We are now validating these results with real time
PCR quantification.
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Searching for Gas41 molecular interactors
We performed a search of Gas41 molecular interactors
in collaboration with Anna Chelstowska (I.B.B.,
Polish Academy of Sciences) by using the yeast twohybrid
system. Gas41 has been used as a bait to screen
pre-transformed library of the human fetal brain. The
screening was performed following the yeast mating
protocol, which improves the sensitivity and the reliability
of the results. The level of stringency was
adjusted by varying the number of the reporter genes
in the primary selection, and this enabled us to identify
weak and strong interactors of Gas41.
Among putative interactors were N-myc, FERM
D4A and RNF8. These proteins were able to interact
also with Yaf9, suggesting that the interaction was
mediated by a structurally conserved domain, possibly
the N-terminal one. N-myc was certainly the
most interesting interactor. The proteins of this
family are very well known transcription factors containing
helix-loop-helix-zipper domains. They are
involved in the control of cell proliferation, differentiation
and apoptosis. Recent data suggest that their
action is mediated by chromatin remodelling complexes.
Each interaction will be confirmed in vitro by
biochemical methods (pull-down, co-immunoprecipitation),
and in mammalian cells by co-transfection
experiments. Validation of the N-myc interaction
was obtained with Gas41 attached to sepharose that
could pull-down both N-myc present in neuroblastoma
cell extract and purified recombinant C-myc.
Selected publications
Del Vescovo V, Casagrande V, Bianchi MM, Piccinni
E, Frontali L, Militti C, Fardeau V, Devaux F, Di Sanza
C, Presutti C, Negri R. Role of Hog1 and Yaf9 in the
transcriptional response of Saccharomyces cerevisiae to
caesium chloride. Physiol Genomics 2008, 33:110-20.