Novel Cell Intrinsic and Extrinsic Mechanisms of X-chromosome Inactivation

Abstract

X-chromosome inactivation equalizes X-linked gene expression between XX female and XY male therian mammals by silencing gene transcription from one X chromosome in early female embryos. X-inactivation is a paradigm of epigenetic transcriptional regulation because two genetically equivalent chromosomes are transcriptionally differentiated and maintain these transcriptional states through many cell divisions. Mice undergo two distinct forms of X-inactivation: imprinted and random. Imprinted X-inactivation results in the silencing of genes on the paternal X chromosome in preimplantation female embryos. Notably, imprinted X-inactivation is a paradigm of transgenerational epigenetic regulation due to its stable parent-of-origin pattern of inactivation of the paternal X chromosome. In this body of work, I discovered specific functions for core Polycomb repressive complex 2 (PRC2) components EED and EZH1/2 in mouse imprinted X-inactivation; a role for inhibition of GSK-3 proteins, which mediate intracellular signaling, in X-inactivation erosion of human embryonic stem cells (hESCs); and defined distinct requirements for Xist RNA versus Xist DNA in X-inactivation.

PRC2 is a protein complex that deposits the histone H3 lysine 27 trimethyl (H3K27me3) chromatin modification that is associated with transcriptional silencing. I identified a role for oocyte-derived (maternal) PRC2 protein EED in preventing inactivation of the maternal X chromosome during imprinted X-chromosome inactivation in mice. I also demonstrate that loss of other PRC2 core proteins, EZH1 and EZH2, in the oocyte results in milder defects in X-inactivation in the embryo, suggesting a role for EED in transcriptional silencing independent of the PRC2 complex.

Unlike mice, all cells in the early female human embryo appear to undergo random X-inactivation, which results in the inactivation of either the maternally or paternally inherited X chromosome in individual cells. I helped identify lithium chloride and other inhibitors of glycogen synthase kinase 3 (GSK-3) proteins in hESC culture media as a cause of X-inactivation erosion via repression of the X-inactivation regulatory long-noncoding RNA XIST. I also discovered that GSK-3 inhibition can repress Xist in mouse embryonic stem cells (mESCs), potentially via the activation of Wnt signaling. GSK-3 inhibition is a new mechanism by which Xist can be regulated and suggests that extracellular signaling can regulate X-inactivation, which is conventionally thought to be regulated cell autonomously. My findings in this study also inform the culture of hESCs.

My thesis work also interrogates the role of Xist RNA versus Xist DNA in X-inactivation in mice. X-inactivation has long been thought to be controlled by Xist RNA, which is expressed solely from the inactive X chromosome and is thought to trigger gene silencing by recruiting protein complexes to the inactive X chromosome. To distinguish a role for Xist RNA from that of the Xist genomic locus, I ablated Xist in female mouse trophoblast stem cells (TSCs), which normally maintain imprinted X-inactivation of the paternal X chromosome. In mouse TSCs devoid of the PRC2 component EED, Xist RNA is not expressed. Despite the absence of Xist RNA, most paternal X-linked genes remain silenced. By contrast, I found that deletion of the Xist genomic loss resulted in de-repression of most paternal X-linked genes. My findings suggest the Xist locus silences X-linked genes by mechanisms other than via Xist RNA.

Together, this work identifies novel intra- and extracellular factors and mechanisms underlying X-inactivation in mice and humans.