Abstract
BackgroundX chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells. Despite identification of the crucial long non-coding RNA Xist and involvement of specific chromatin modifiers in the establishment and maintenance of the heterochromatin of the inactive X chromosome (Xi), interference with known pathways only partially reactivates the Xi once silencing has been established. Here, we studied ATF7IP (MCAF1), a protein previously characterized to coordinate DNA methylation and histone H3K9 methylation through interactions with the methyl-DNA binding protein MBD1 and the histone H3K9 methyltransferase SETDB1, as a candidate maintenance factor of the Xi.ResultsWe found that siRNA-mediated knockdown of Atf7ip in mouse embryonic fibroblasts (MEFs) induces the activation of silenced reporter genes on the Xi in a low number of cells. Additional inhibition of two pathways known to contribute to Xi maintenance, DNA methylation and Xist RNA coating of the X chromosome, strongly increased the number of cells expressing Xi-linked genes upon Atf7ip knockdown. Despite its functional importance in Xi maintenance, ATF7IP does not accumulate on the Xi in MEFs or differentiating mouse embryonic stem cells. However, we found that depletion of two known repressive biochemical interactors of ATF7IP, MBD1 and SETDB1, but not of other unrelated H3K9 methyltransferases, also induces the activation of an Xi-linked reporter in MEFs.ConclusionsTogether, these data indicate that Atf7ip acts in a synergistic fashion with DNA methylation and Xist RNA to maintain the silent state of the Xi in somatic cells, and that Mbd1 and Setdb1, similar to Atf7ip, play a functional role in Xi silencing. We therefore propose that ATF7IP links DNA methylation on the Xi to SETDB1-mediated H3K9 trimethylation via its interaction with MBD1, and that this function is a crucial feature of the stable silencing of the Xi in female mammalian cells.
Highlights
X chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells
We reveal this function of Atf7ip, Mbd1, and Setdb1 in a ‘sensitized’ approach, where we add low concentrations of 5-aza-2’-dC to Xi-reactivation assays with the rationale of destabilizing DNA methylation and XCI, so that interference with candidate gene function would increase rates of Xi-reactivation in somatic cells
Heard and colleagues found ATF7IP (MCAF1) and its homolog ATF7IP2 (MCAF2) as one of 20 high-confidence proteins interacting with CDYL in both undifferentiated and differentiating mouse embryonic stem cells (ESCs) [27], leading us to test the hypothesis that ATF7IP, a protein known to link the DNA methylation and H3K9 methylation pathways in autosomal gene silencing contexts, could function in XCI
Summary
X chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells. Random XCI is initiated in early embryonic development in pluripotent epiblast cells shortly after the blastocyst implants. The initiation of XCI can be recapitulated in differentiating female mouse embryonic stem cells (ESCs). Despite its extreme stability in differentiated cells in vivo, the reactivation of the Xi can be achieved experimentally by induction of the pluripotent state in somatic cells either through transcription factor-induced reprogramming to induced pluripotent stem cells (iPSCs), cell fusion with ESCs, or somatic cell nuclear transfer [6,7,8]
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