The Mbd1-Atf7ip-Setdb1 pathway contributes to the maintenance of X chromosome inactivation

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.

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression

“…2A and Dataset S2). The modest level of reactivation with down-regulation of a single target was consistent with results in other screens for X-chromosome reactivation (18)(19)(20)(21), indicating that several separate mechanisms are necessary for the tight transcriptional repression of the MeCP2 locus. To determine whether we could increase the level of reactivation by inactivating multiple targets, we combined shRNAs with 5-AZA in a dose that by itself did not cause appreciable reporter reactivation.…”

“…To determine whether the genes in our screen reactivated other genes on the X chromosome, we used a mouse cell line carrying the CMV-luciferase reporter inserted on the Xi (18). We observed that, when combined with low-dose 5-AZA, each of the hairpins that reactivated the MeCP2 reporter also reactivated the CMV-luciferase reporter locus on the Xi (18) (Fig. 2D and Dataset S2), indicating that the hits we have identified are not selective for reactivation of the MeCP2 locus but can instead derepress transcription at other loci on the Xi.…”

X-chromosome inactivation: new insights into cis and trans regulation

“…CDYL also associates with the H3K9 methyltransferases G9a and SETDB1, thus may promote the propagation of the H3K9me2 mark [67,70]. SETDB1 itself is involved in a pathway that contributes to the maintenance of XCI in somatic cells and that might bridge DNA methylation and H3K9 methylation in XCI, through the involvement of a methyl-DNA binding protein, MBD1 [70]. Clearly the discovery and purification of inactive X chromosome proteins and their partners will provide a handle with which to define the molecular mechanisms underlying XCI.…”

The making of a Barr body: the mosaic of factors that eXIST on the mammalian inactive X chromosome

“…H3K9me3 is strongly associated with constitutive heterochromatin and promoting the recruitment of DNA Methyltransferases (DNMTs) that lead to DNA methylation early in development ( Figure 4D) (Russo et al 2013). Studies in mice suggest that H3K9me3 is generated along the Xi by SETDB1 monomethylating H3K9me2 and knockdown of Setdb1 promotes reporter reactivation on the Xi (Minkovsky et al 2014). H3K9me3 plays a critical role in establishing the heterochromatin of Domain 2 on the Xi, in part through the recruitment of heterochromatin protein 1 (HP1) (Lachner et al 2001).…”

“…This leads to H3K9me3 and H4K20me3 enrichment as well as H3K9me2 and H4K20me1 depletion in Domain 2 heterochromatin. HP1 binds H3K9me3, which is needed for the recruitment of SUV420H2 as well as the enzyme DNMT3b, which methylates the CpG island promoters along the Xi ( Figure 4) (Chan et al 2011;Kapoor-Vazirani and Vertino 2014;Minkovsky et al 2014).…”