The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation - PubMed (original) (raw)

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

Sundeep Kalantry et al. Nat Cell Biol. 2006 Feb.

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 expression. In mouse embryos that are mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues. 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 lineage. 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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Figures

Figure 1

Figure 1

The paternal X-chromosome (Xp) is active only in differentiating _Eed_−/− trophoblast stem (TS) cells. An Xp-linked GFP transgene is used as a reporter of X-linked gene activity and nuclei are stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). (a) Wild-type (WT) TS cells undergo imprinted XCI of the Xp, as indicated by a complete lack of Xp-GFP expression in all cells, including after differentiation. _Eed_−/− TS colonies contain cells with an active Xp located preferentially at the periphery of the colonies, where differentiated cells are found. (b) Immunofluorescence (IF) detection of Cdx2, a marker of undifferentiated trophectoderm cells, in cultured _Eed_−/− TS cells. Cdx2 is downregulated in mutant cells harboring an active Xp, as indicated by GFP expression, indicating that these cells are differentiated. (c) RT-PCR analysis of male and female WT and _Eed_−/− TS cells for markers of undifferentiated and differentiated trophoblast cells. Cdx2, Eomes (Eomesodermin), and Fgf receptor 2 (FgfR2), all markers of undifferentiated trophectoderm cells, are expressed in all four cell lines examined. Hand1, a marker of intermediate, non-giant differentiated trophoblast cells is also expressed in all cell lines. Pl1 (Placental lactogen 1), a marker of trophoblast giant cells, is absent only in female _Eed_−/− TS cells. Thus, _Eed_−/− female, but not male, TS cells are blocked from terminal differentiation into giant cells, consistent with reactivation of the Xp during initial differentiation of the female mutant TS cells resulting in a block to their further differentiation. (d) Trophoblast (TB) cells located on the periphery of WT blastocyst outgrowths lack Xp-activity, as indicated by Xp-GFP expression, while TB cells in _Eed_−/− female blastocyst outgrowths harbor an active Xp.

Figure 2

Figure 2

All features of the Xi-heterochromatin are absent in _Eed_−/− female TS cells. (a) IF detection of the Polycomb repressive complex 2 (PRC2) proteins Ezh2, Su(z)12, the PRC2-mediated histone modification tri-methyl lysine 27 of histone H3 (H3-3mK27), the PRC1 proteins Cbx2 and Phc2, PRC1-like mediated histone modification ubiquitylated-H2A (Ub-H2A), Pr-Set7 mediated histone modification mono-methyl lysine 20 of histone H4 (H4-1mK20), and the histone variant macroH2A. Left three rows, WT female TS cells; right three rows, _Eed_−/− female TS cells. Middle panels in both WT and _Eed_−/− TS cell columns are Eed immunostains; right panels are nuclei stained with DAPI. All proteins or epigenetic marks that colocalize with Eed on the inactive-X in WT TS cells, and are absent in all _Eed_−/− TS cells.

Figure 3

Figure 3

Xist RNA fails to coat the Xp in all _Eed_−/− TS cells. (a) IF-FISH detection of Eed (purple) and Xist RNA (red) in wild-type (WT) and _Eed_−/− TS cells. In WT TS cells, Xist (red) and Eed (purple) colocalize on the Xi in the nucleus (blue); _Eed_−/− TS cells lack Xist RNA coating of the Xi. (b) Trophoblast (TB) cells in cultured _Eed_−/− blastocysts also show lack of Xist RNA accumulation onto the Xi. WT blastocyst outgrowths harbor TB giant cells characterized by larger nuclei with endoreduplicated genomes and multiple inactive-Xs, as marked by multiple Xist foci. _Eed_−/− female embryos do not differentiate TB giant cells, due to X-inactivation defect in diploid TB cells. Inner cell mass-derived cells in _Eed_−/− blastocysts, however, do display Xist RNA accumlation onto the Xi. (c) RT-PCR analysis of Xist and Tsix RNAs in WT and _Eed_−/− TS cells. Xist is expressed in _Eed_−/− TS cells, but its steady-state levels are decreased compared to WT TS cells. Tsix is not detectable in both WT and _Eed_−/− TS cells. (d) Real-time RT-PCR quantitation of Xist RNA in _Eed_−/− TS cells relative to WT TS cells. Male mouse embryonic fibroblast cells serve as control not expressing Xist RNA.

Figure 4

Figure 4

Absence of an epigenetic hallmark of active chromatin, histone H3-di-methyl lysine 4 (H3-2mK4), from the paternal X-chromosome (Xp) in _Eed_−/− TS cells. (a) IF detection of H3-2mK4 (green) and FISH detection of Xist RNA (red) and Xist merged with H3-2mK4 in a representative WT female TS cell nucleus. The Xi, as marked by Xist RNA accumulation, is devoid of H3-2mK4 in all WT TS cells. Xist RNA does not coat the Xi in _Eed_−/− female TS cells but is detected as a pinpoint. In most mutant TS cells, the pinpoint Xist RNA signal falls within a hole devoid of H3-2mK4. Nuclei are stained blue with DAPI. (b) Similar percentages of cells have an active Xp, as assayed by Xp-GFP expression, and an Xp that overlaps with H3-2mK4 staining, suggesting that trophoblast cells with an active Xp also harbor H3-2mK4 on that chromosome. (c) H3-2mK4 IF and FISH detection of the Xp-GFP RNA in _Eed_−/− female TS cells. (d) 95% of Xp-GFP expressing _Eed_−/− TS cells also have H3-2mK4 staining of the Xp. Only differentiating _Eed_−/− TS cells reactivate the Xp (Fig. 1) and these cells no longer exclude marks correlated with transcriptional activity.

Figure 5

Figure 5

Lack of XCI defects in the trophectoderm-derived undifferentiated extra-embryonic ectoderm and the differentiated derivatives of the primitive endoderm in _Eed_−/− embryos. (a) WT embryos do not display any Xp-activity in the extra-embryonic tissues of the embryo, due to imprinted XCI of the Xp in these cells. The epiblast (embryo proper, red arrow) undergoes random XCI, resulting in a mosaicism of X-chromosome activity; in some cells the maternal-X is active and in some cells the Xp is active, as indicated by Xp-GFP expression. E6.2 _Eed_−/− female embryos reactivate the Xp only in differentiating trophoblast (TB) cells in the ectoplacental cone (EPC; the proximal end of the embryo, yellow arrow). Undifferentiated extra-embryonic ectoderm (ExE, white arrow), a source of trophoblast stem (TS) cells and precursors of differentiated trophoblast cells, and visceral endoderm layer (VE, light blue arrow) are devoid of Xp-activity in _Eed_−/− embryos. (b) Absence of PRC2 enrichment on the inactive-Xp in female primitive endoderm-derived (Endo) cell lines. (c) Visceral endoderm cells isolated from E6.5 mouse embryos also accumulate Xist RNA but not Eed on the inactive-X (Xi). Nuclei are stained with DAPI.

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