The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation

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Abstract

The Polycomb group (PcG) encodes an evolutionarily conserved set of chromatin-modifying proteins that are thought to maintain cellular transcriptional memory by stably silencing gene expression1. In mouse embryos that are mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues2. Eed is a component of a histone-methyltransferase complex that is thought to contribute to stable silencing in undifferentiated cells due to its enrichment on the inactive X-chromosome in cells of the early mouse embryo and in stem cells of the extra-embryonic trophectoderm lineage3,4,5,6,7,8. Here, we demonstrate that the inactive X-chromosome in Eed−/− trophoblast stem cells and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed−/− embryos remain transcriptionally silent, despite lacking the PcG-mediated histone modifications that normally characterize the facultative heterochromatin of the inactive X-chromosome. Whereas undifferentiated Eed−/− trophoblast stem cells maintained XCI, reactivation of the inactive X-chromosome occurred when these cells were differentiated. These results indicate that PcG complexes are not necessary to maintain transcriptional silencing of the inactive X-chromosome in undifferentiated stem cells. Instead, PcG proteins seem to propagate cellular memory by preventing transcriptional activation of facultative heterochromatin during differentiation.

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Figure 1: The paternal X-chromosome is active only in differentiating Eed−/− trophoblast stem cells.
Figure 2: All features of the inactive X-chromosome heterochromatin are absent in Eed−/− female trophoblast stem cells.
Figure 3: Xist RNA fails to coat the paternal X-chromosome in all Eed−/− trophoblast stem cells.
Figure 4: Absence of an epigenetic hallmark of active chromatin, H3-2mK4, from the paternal X-chromosome in Eed−/− trophoblast stem cells.
Figure 5: Lack of X-chromosome inactivation defects in the trophectoderm-derived undifferentiated extra-embryonic ectoderm and the differentiated derivatives of the primitive endoderm in Eed−/− embryos.

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Acknowledgements

We are grateful to T. Jenuwein for sharing the anti-H3-3mK27 antibody; and to K. Worringer, S. Mlynarczyk-Evans and A. Anderson for supplying Xist, Hprt, Mecp2 and Pgk1 FISH templates and probes. We also acknowledge S. Mlynarczyk-Evnas for technical advice on FISH. S.K. is a recipient of an American Cancer Society postdoctoral fellowship. This work was funded by a National Institutes of Health grant to T.M.

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Correspondence to Terry Magnuson.

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Supplementary Figures S1, S2, S3 and S4 plus Supplementary Methods (PDF 899 kb)

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