Review
X-chromosome epigenetic reprogramming in pluripotent stem cells via noncoding genes

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Abstract

Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome. Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell differentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation center. This review article will discuss the latest advances in our understanding at the molecular level. Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is characterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks on the X-chromosome indicate whether human pluripotent stem cells are developmentally ‘naïve’, with characteristics of the pluripotent ground state. X-chromosome status and determination thereof via noncoding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem cells, an important consideration given their enormous potential for stem cell therapy.

Introduction

This review article will discuss the tight linkage between X-chromosome and stem cell reprogramming. Recent studies have shown that this linkage is mediated by pluripotency factors acting specifically on noncoding genes of the X-inactivation center (Xic) to initiate or reverse X-chromosome inactivation (XCI), the mechanism of dosage compensation in mammals which leads to transcriptional inactivation of one X-chromosome in the female. XCI provides a classic model for noncoding RNA (ncRNA)-mediated epigenetic regulation [1], [2], [3]. These ncRNAs are located at the Xic, a regulatory hub that mediates the stepwise formation of Xi heterochromatin [4]. The onset of XCI corresponds with expression of the 17-kb noncoding Xist RNA, which coats the entire inactive X (Xi) chromosome in cis [5], [6], [7], [8], [9], [10], [11]. Xist mediates facultative heterochromatin on the Xi through recruitment and interaction with Polycomb group proteins [12], marking the Xi with histone H3 lysine 27 trimethylation (H3K27me3) [13], [14], [15]. Xist expression is regulated by three other ncRNAs, with two functioning in the activation of Xist (RepA, Jpx) [12], [16], [17] and one functioning to antagonize its activation (Tsix) [18], [19], [20].

Although this review will not focus on imprinted X-chromosome inactivation, it should be briefly mentioned that XCI can be subject to parental imprinting in marsupial mammals and also in the extraembryonic lineages of some eutherian mammals (e.g., mouse, cow) [21], [22]. Imprinted XCI occurs on the paternal X-chromosome and is believed to be the ancestral form of mammalian dosage compensation. In mice, the imprinted form of XCI is observed first during development in all cells, but persists only in the extraembryonic tissues after embryonic day 4.5, when imprint erasure and X-reactivation occur in the epiblast lineage [23], [24], [25], [26]. Among ncRNAs involved in “random” XCI, Xist and Tsix are thus far the only ones known to also participate in imprinted XCI. Embryos lacking Tsix cannot protect the maternal X-chromosome from silencing [20], [27], and those lacking Xist cannot initiate genic silencing on the paternal X [10], [25].

Following reactivation of the paternal X-chromosome, cells of the epiblast lineage undergo random XCI and give rise to the embryo proper. From mouse and human embryos, it is possible to derive cells from this lineage and generate embryonic stem (ES) cells, a pluripotent cell type capable of differentiating into all three germ lineages (ectoderm, mesoderm, endoderm). ES cells have provided a valuable ex vivo system for the study of epigenetic reprogramming and the role of XCI and ncRNAs during cell differentiation [1], [2], [3], [28]. With the possibility of creating induced pluripotent stem (iPS) cells from adult somatic cells [29], [30] has come the opportunity to study how and whether reprogramming into pluripotent stem cells is accompanied by X-reactivation. These studies have shown that events on the X-chromosome and stem cell fate are indeed intimately connected. Below, we will focus on events surrounding cell differentiation and de-differentiation and the fate of the X-chromosome in ES and iPS cells, specifically those involving noncoding genes.

Section snippets

Mouse ES cells

For random XCI studies, mouse ES cells [31] have served as a powerful model system and enabled elucidation of function for many ncRNAs during this process. In undifferentiated female mES cells where parental epigenetic marks have been erased to be reprogrammed, both Xs remain active with very low levels of Xist expression. Cell differentiation then triggers XCI, initiated with Xist RNA upregulation on the future Xi. Although how Xist is regulated has yet to be fully understood, many studies

Human ES cells

Assessing XIST ncRNA and XCI status in hES cells provides a measure of their epigenetic stability, which is an important consideration for their potential applications in regenerative medicine. Existing hES cell lines exhibit diverse patterns of XIST expression, indicative of both pre- and post-XCI states [65], [66], [67], [68], [69], [70]. Because of similarities between mEpiSCs and hES cells (morphology, Activin/Nodal signaling for pluripotency, bFGF growth requirements), it was hypothesized

Conclusions

The review presented here recapitulates how XCI is achieved by noncoding genes (Xist, Tsix, Xite, RepA, and Jpx) in pluripotent stem cells and provides evidence for a tight linkage between these noncoding elements and core pluripotency factors in the control of XCI. Because hES cells can be isolated in a pre-XCI state, the mechanisms of XCI between human and mouse might be more similar than previously thought. Deciphering the molecular mechanisms underlying X-chromosome reprogramming may yield

Acknowledgements

We thank all laboratory members for valuable discussions. D.H.K. is supported by a Damon Runyon Cancer Research Foundation Fellowship (DRG-#2027-09) and the Beckman Fellows Program at Caltech, Y.J. by a Korean Research Foundation grant (C00069) and a Discovery grant from MGH ECOR, M.C.A. by NIH-T32CA009216, and J.T.L. by NIH-GM58839. J.T.L is an Investigator of the Howard Hughes Medical Institute.

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