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Comparative Study
. 2004 Dec 20;167(6):1025-35.
doi: 10.1083/jcb.200409026. Epub 2004 Dec 13.

Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome

Kathrin Plath  1 , Dale Talbot, Karien M Hamer, Arie P Otte, Thomas P Yang, Rudolf Jaenisch, Barbara Panning
Affiliations
Comparative Study

Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome

Kathrin Plath et al. J Cell Biol. .

Abstract

Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.

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Figures

Figure 1.
Figure 1.
Localization of mPRC1 proteins in somatic cells. (a–c) MEFs were immunostained for Bmi-1, Cbx2, or Phc2 (second column) in combination with FISH for Xist RNA to detect the Xi (third column). DAPI delineates the nucleus (first column), and the merge presents Xist RNA in green and mPRC1 proteins in red (fourth column). (d) Transformed MEFs were stained for Phc1 (second column) and macroH2A (third column), to mark the Xi (Costanzi and Pehrson, 1998). Nuclei are visualized by DAPI staining (first column). The merge shows mPh1 in green and macroH2A in red (fourth column).
Figure 2.
Figure 2.
mPRC1 protein localization in TS cells. Immunostaining for Eed (second column) and mPRC1 proteins (third column) in female TS cells. (a–d) DAPI staining (first column) was used to mark the nuclei and the merge (fourth column) represents Eed in green and Bmi-1 (a), Cbx2 (b), Phc2 (c), or Phc1 (d) protein in red. (e–g) Bmi-1 shows mitotically stable association with the Xi, as enrichment of Bmi-1 can be detected on the Eed-marked Xi in prophase (e), metaphase (f), and anaphase (g). Phc1, Phc2, and Cbx2 also exhibit mitotically stable Xi enrichment (unpublished data).
Figure 3.
Figure 3.
Immunolocalization of human PRC1 proteins in 293 cells. (a) 293 cells were stained for H3-3mK27 (third column) in combination with FISH for XIST RNA to detect the Xi (second column). Nuclei were stained with DAPI (first column) and merged image (fourth column) consists of XIST RNA in green and H3-3mK27 in red. 293 cells contain two Xis and virtually all cells showed Xi accumulation of H3-3mK27 (unpublished data). (b) Immunostaining for H3-3mK27 (third column) and hPRC1 proteins (second column) in 293 cells. DAPI staining (first column) was used to mark the nuclei and the merge (fourth column) represents H3-3mK27 in red and hPRC1 proteins in green. The fraction of cells with an H3-3mK27-marked Xi that also exhibited Xi enrichment for each mPRC1 protein assayed is indicated on the right. All percentages are based on counts of >75 cells.
Figure 4.
Figure 4.
Localization of mPRC1 proteins in cells lacking Xis t RNA on the Xi. (a and b) Detection of Xist RNA (second column) and H3-3mK27 (third column) in DAPI stained (first column) MEFs containing a conditional Xist allele on the Xi. The merge (fourth column) represents the overlay of H3-3mK27 (red) and the Xist RNA (green). (a) Most parental Xist+ cells contain two Xis, both of which are characterized by colocalization of Xist RNA and H3-3mK27. (b) After Cre-mediated deletion of Xist from the Xi, generating Xist− cells, no enrichment of H3-3mK27 or Xist RNA is detected on the Xi. (c) The proportion of cells with Xi or Xi-like enrichment of Bmi-1, Cbx2, Phc2, and H3-3mK27. In Xist+ cells the Xi accumulation of mPRC1 proteins or H3-3mK27 was confirmed by overlap with the area marked by Xist RNA. After addition of Cre, Xist− cells no longer expressed Xist RNA, and cells were scored for a pattern of mPRC1 or H3-3mK27 staining that overlapped with the DAPI-intense Barr body, which delineates the Xi. Percentages and standard errors are calculated from counts of >200 cells from three experiments.
Figure 5.
Figure 5.
Localization of mPRC1 proteins in undifferentiated male ES cells which ectopically express Xis t. (a) Xist expression was induced in undifferentiated male ES cells for 0 or 24 h and distribution of Xist RNA and Eed determined by immuno-FISH. The graph depicts the percentage of cells with an Xist RNA-coated chromosome that displayed overlapping accumulation of Eed. Percentages are based from counts of ∼100 cells each from three independent experiments. (b) Graph displaying the proportion of cells that display Phc1, Phc2, Bmi-1, or Cbx2 enrichment on the Eed-marked Xi, at 0 and 24 h after induction. Percentages are calculated from six independent experiments in each of which ∼100 cells with an Eed-marked Xi were counted. (c) The graph displays the fraction of cells that exhibit Xi enrichment of Eed or Phc1 on the Xist RNA-coated chromosome 24 h after induction of wild-type or A-repeat delete Xist cDNA expression in undifferentiated ES cells. The proportion of cells in which Xist expression was induced varied from 30 to 70%, in four experiments. Percentages are based on counts of >150 cells that exhibited Xist RNA coating. In all instances error bars indicate standard errors.
Figure 6.
Figure 6.
Kinetics of Xi enrichment of mPRC1 proteins in differentiating ES cells. The proportion of differentiating female ES cells exhibiting Xi enrichment for Eed (a), Phc1 (b), Bmi-1 (c), Cbx2 (d), and Phc2 (e) were determined over an 11 d time course. At each time point the percentage of Xist RNA-coated Xis that exhibited enrichment for the PcG protein indicated was calculated. At day 0 >100 cells were assayed and Xi enrichment of Xist or mPRC1 proteins was not detected. At each time point after day 0, percentages are based on counts of between 100 and 750 nuclei that exhibited Xist RNA coating of the Xi.
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