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C. Turano - Roles of ERp57 in DNA repair and in the regulation of gene expression
(ChIP) on intact HeLa, Raji and M14 cells. Cloning
and sequencing of the DNA immunoprecipitated
from ChIP experiments in HeLa cells allowed the
identification of 37 sites on DNA as targets for
ERp57, most of which were validated by PCR in all
three cell types examined. Of these 37 sites, 15 were
introns of known genes, 5 were on the 5’ or 3’ flanking
regions of genes, and the others were in intergenic
regions. The identified genes were involved in
cellular regulatory processess (such as DNA repair,
cell adhesion or vescicular traffic) rather that in
metabolic pathways. In HeLa cells, three gene targets
were present in DNase hypersensitive sites,
which are most likely regulatory sites of gene
expression. Among the genes targeted by ERp57
are POLD1 (the catalytic subunit of the DNA polymerase
δ) and MSH6, both involved in DNA repair,
and LRBA, involved in response to lipopolysaccharide.
The products of these genes are proteins
whose functions are compatible with a response to
cellular stress. Considering that ERp57 is a stress
response protein itself, it is likely that ERp57 acts in
part by regulating the expression of proteins
involved in such response.
The expression of one of the MSH6 gene was measured
in conditions of a decreased amount of ERp57.
This was achieved by RNA interference of ERp57,
which caused a significant decrease in MSH6 expression
both in HeLa and M14 cells.
The intervention of ERp57 in gene expression regulation
was confirmed by studying some STAT3-dependent
genes in M14 cells. In five of these genes (A2M,
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CRP, CDKN1A, CDC25A, MMP9), ChIP experiments
demonstrated that ERp57 was bound directly to DNA
in proximity of STAT3. The expression of the same
five genes was clearly decreased upon siRNA-ERp57
treatment of the cells. Inhibitors of ERp57 appeared
to hinder the binding in vitro of STAT3 to its consensus
sequence. Taken on the whole, these data provide
conclusive evidence of an involvement of nuclear
ERp57 in transcription regulation.
At present, we are also investigating the function of
ERp57 on the cell surface, where it seems to involved
in the process of hormone- or growth factor-receptor
interactions, with a possible intervention in internalization
and/or nuclear import phenomena.
Selected publications
Chichiarelli S, Ferraro A, Altieri F, Eufemi M,
Coppari S, Grillo C, Arcangeli V, Turano C. The
stress protein ERp57/GRP58 binds specific DNA
sequences in HeLa cells. J Cell Physiol. 2007,
210:343-51.
Grillo C, D'Ambrosio C, Consalvi V, Chiaraluce R,
Scaloni A, Maceroni M, Eufemi M, Altieri F. DNAbinding
activity of the ERp57 C-terminal domain is
related to a redox-dependent conformational change.
J Biol Chem. 2007, 282:10299-310.
Gaucci E, Chichiarelli S, Grillo C, Vecchio ED,
Eufemi M, Turano C. The binding of antibiotics to
ERp57/GRP58. J Antibiot. (Tokyo) 2008, 61:400-2.