The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation

Abstract

The Polycomb group (PcG) proteins are thought to silence gene expression by modifying chromatin. The Polycomb repressive complex 2 (PRC2) plays an essential role in mammalian X-chromosome inactivation (XCI), a model system to investigate heritable gene silencing. In the mouse, two different forms of XCI occur. In the preimplantation embryo, all cells undergo imprinted inactivation of the paternal X-chromosome (Xp). During the peri-implantation period, cells destined to give rise to the embryo proper erase the imprint and randomly inactivate either the maternal X-chromosome or the Xp; extraembryonic cells, on the other hand, maintain imprinted XCI of the Xp. PRC2 proteins are enriched on the inactive-X during early stages of both imprinted and random XCI. It is therefore thought that PRC2 contributes to the initiation of XCI. Mouse embryos lacking the essential PRC2 component EED harbor defects in the maintenance of imprinted XCI in differentiating trophoblast cells. Assessment of PRC2 requirement in the initiation of XCI, however, has been hindered by the presence of maternally derived proteins in the early embryo. Here we show that Eed-/- embryos initiate and maintain random XCI despite lacking any functional EED protein prior to the initiation of random XCI. Thus, despite being enriched on the inactive X-chromosome, PcGs appear to be dispensable for the initiation and maintenance of random XCI. These results highlight the lineage- and differentiation state-specific requirements for PcGs in XCI and argue against PcG function in the formation of the facultative heterochromatin of the inactive X-chromosome.

Figure 1. Absence of EED Protein in Eed ^−/− Embryos at E4.5

(A) IF detection of EED protein in an early E3.5 blastocyst stage embryo showing Xi enrichment of EED in all cells. (B) IF detection in a hatched, late-stage E3.5 blastocyst shows accumulation of EED on the Xi in trophoblast cells, an extraembryonic cell type that maintains imprinted XCI of the Xp but lacking Xi enrichment in ICM cells. The ICM cells at this stage of development undergo erasure of the imprint that ensures preferential transcriptional inactivation of the Xp. (C) IF detection of Xi-enriched EED protein in a WT E4.5 embryo. The Xi is coated with the Xist RNA, detected by FISH. DAPI staining detects nuclei. (D) IF-FISH staining of an E4.5 female embryo showing an absence of EED protein. The EED antibody staining seen is background and does not overlap with the Xi, as marked by Xist RNA coating.

Figure 2. Absence of H3-3mK27 in Eed ^−/− Embryos at E4.5

(A) IF-FISH detection shows Xi enrichment of EED protein and H3-3mK27 in a WT E4.5 embryo. The Xi is marked by Xist RNA coating, detected by FISH. DAPI staining detects nuclei. (B) An E4.5 female embryo that is devoid of EED and H3-3mK27. The EED antibody staining seen is background and does not overlap with the Xi, as denoted by Xist RNA coating.

Figure 3. Absence of EED Activity prior to the Initiation of Random XCI in Eed ^−/− Embryos

(A) IF detection of H3-3mK27 in a WT E4.5 embryo shows H3-3mK27 enrichment on the Xi in all trophectodermal cells, but which is largely absent in the ICM/epiblast cells indicating that these cells have not undergone random XCI. The selective expression of the paternal X-linked GFP transgene (Xp-GFP) in the ICM lineage indicates that these cells have erased the imprint that ensures imprinted XCI of the Xp. Whereas the ICM has erased the imprint, the trophectoderm has stably maintained imprinted XCI of the Xp, and hence is negative for Xp-GFP. DAPI staining detects the nuclei. (B) IF staining showing a lack of histone H3-3mK27 accumulation on the Xi in all cells at E4.5, indicating that EED activity is absent in mutant embryos prior to the initiation of random XCI.

Figure 4. Enrichment of H3-3mK27 on the Xi during Initiation of Random XCI

(A) IF detection in a WT E5.5 embryo showing Xi accumulation of H3-3mK27 in all cells of both the extraembryonic and embryonic lineages, i.e., the trophectoderm-derived extraembryonic ectoderm and the epiblast, which undergo imprinted and random XCI, respectively. (B) Eed ^−/− embryos lack H3-3mK27.

Figure 5. Absence of EED Protein in Epiblast Cells of E6.5 Eed ^−/− Embryos

IF-FISH detection shows that whereas isolated E6.5 WT epiblast cells accumulate EED on the Xi, denoted by Xist RNA coating, epiblast cells from Eed ^−/− female embryos lack such enrichment.

Figure 6. Lack of Defects in Random XCI in Post–Implantation Stage Eed ^−/− Embryos

Both WT and Eed ^−/− embryos contain on their Xp a GFP transgene and a mutation in the Tsix gene (XX^{GFP; ΔTsix} and XX^{GFP; ΔTsix}; Eed ^−/−, respectively). The extraembryonic ectoderm (ExE) and its derivatives maintain imprinted XCI of the Xp that is established in all cells of the preimplantation embryo, while the embryonic epiblast lineage undergoes erasure of the imprint at the late blastocyst stage resulting in reactivation of the Xp. This is then followed by random inactivation of either the paternal or maternal X-chromosome during postimplantation development. The Tsix mutation on the paternal X-chromosome results in its preferential inactivation in the epiblast. GFP fluorescence in the epiblast or epiblast derivatives of E6.5 and E7.5 embryos is due to residual Xp activity after its reactivation at the late blastocyst stage. By E8.5 when random XCI is complete, epiblast derivatives of both XX^{GFP; ΔTsix} and XX^{GFP; ΔTsix}; Eed ^−/− embryos lack expression of the Xp-GFP transgene, indicating that Eed is not required for the maintenance of random XCI. Dashed lines delineate border between embryonic and extraembryonic compartments of the embryo; TB, trophoblast cells. XX^{GFP; ΔTsix} embryos exhibit a complete absence of Xp-GFP fluorescence in the extraembryonic compartment. XX^{GFP; ΔTsix}; Eed ^−/− embryos, however, harbor increasing numbers of GFP-expressing trophoblast cells, indicating that Tsix is not required for reactivation of the Xp.

Figure 7. Monoallelic Expression of Endogenous X-linked Genes in Epiblast Derivatives of Female E7.5 Eed ^−/− Embryos

FISH detection of Xist, Hprt, Mecp2, and Pgk1 transcripts indicates that endogenous X-linked genes are expressed from one X-chromosome in Eed ^−/− epiblast-derived cells. Epiblast derivatives from both WT and Eed ^−/− E7.5 embryos show similar rates of monoallelic X-linked gene expression (>96%). Hprt, Mecp2, and Pgk1 are invariably expressed from the chromosome other than the one marked by Xist RNA coating, which denotes the Xi. Sixty-eight to 87 nuclei were counted for each sample.