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

Plath K, Talbot D, Hamer KM, Otte AP, Yang TP, Jaenisch R, Panning B - J. Cell Biol. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA. plath@wi.mit.edu

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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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).
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fig1: 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).

Mentions: To determine whether mPRC1 proteins are enriched on the Xi, we analyzed the distribution of Bmi-1, Cbx2, Phc1 and Phc2 in transformed mouse embryo fibroblasts (MEFs), which are tetraploid and contain two Xis. Immunostaining for these mPRC1 proteins was combined with FISH for Xist RNA or immunostaining for the histone variant macroH2A to mark the Xi (Fig. 1, a–d). When tested on MEFs overexpressing mPRC1 proteins, the antibody recognizing Phc2 showed some cross-reactivity for Phc1 and Phc3, whereas the other antibodies exhibited specificity and did not recognize closely related homologues (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200409026/DC1). All four mPRC1 proteins showed diffuse nuclear staining with exclusion from the nucleolus and pericentric heterochromatin and a variable number of speckles, and in a subset of cells these mPRC1 proteins showed Xi enrichment (Table II). Bmi-1 and Cbx2 exhibited Xi enrichment in 12.1% and 13.9% of cells, a proportion similar to that seen when tagged Bmi-1 or Cbx2 were expressed in MEFs (Fig. S2 and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200409026/DC1), suggesting that antibody staining accurately reflects the small proportion of cells with Xi enrichment of these mPRC1 proteins. Phc2 accumulated on the Xi in 3.4% of cells when assayed by immunostaining, whereas tagged Phc2 accumulated on the Xi in 27% of cells (Fig. S2 and Table S1), suggesting that possibility that more cells exhibit Xi enrichment of Phc2 than is indicated by antibody staining. Phc1 exhibited Xi enrichment in <1% of cells by immunostaining and expression of tagged protein, and Phc1 unusual in that in many instances it was detected on only one of the two Xis (unpublished data). In cells assayed for localization of macroH2A and Phc1, both proteins exhibited some overlapping sites of enrichment at regions other than the Xi. Similar results were observed when macroH2A was examined in combination with other mPRC1 proteins (unpublished data), suggesting that localization of mPRC1 proteins and this variant histone may be regulated by related mechanisms. These data demonstrate that Bmi-1, Cbx2, and Phc2 exhibit Xi localization in a significant proportion of cells, indicating a role for mPRC1 proteins in X inactivation.


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

Plath K, Talbot D, Hamer KM, Otte AP, Yang TP, Jaenisch R, Panning B - J. Cell Biol. (2004)

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).
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fig1: 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).
Mentions: To determine whether mPRC1 proteins are enriched on the Xi, we analyzed the distribution of Bmi-1, Cbx2, Phc1 and Phc2 in transformed mouse embryo fibroblasts (MEFs), which are tetraploid and contain two Xis. Immunostaining for these mPRC1 proteins was combined with FISH for Xist RNA or immunostaining for the histone variant macroH2A to mark the Xi (Fig. 1, a–d). When tested on MEFs overexpressing mPRC1 proteins, the antibody recognizing Phc2 showed some cross-reactivity for Phc1 and Phc3, whereas the other antibodies exhibited specificity and did not recognize closely related homologues (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200409026/DC1). All four mPRC1 proteins showed diffuse nuclear staining with exclusion from the nucleolus and pericentric heterochromatin and a variable number of speckles, and in a subset of cells these mPRC1 proteins showed Xi enrichment (Table II). Bmi-1 and Cbx2 exhibited Xi enrichment in 12.1% and 13.9% of cells, a proportion similar to that seen when tagged Bmi-1 or Cbx2 were expressed in MEFs (Fig. S2 and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200409026/DC1), suggesting that antibody staining accurately reflects the small proportion of cells with Xi enrichment of these mPRC1 proteins. Phc2 accumulated on the Xi in 3.4% of cells when assayed by immunostaining, whereas tagged Phc2 accumulated on the Xi in 27% of cells (Fig. S2 and Table S1), suggesting that possibility that more cells exhibit Xi enrichment of Phc2 than is indicated by antibody staining. Phc1 exhibited Xi enrichment in <1% of cells by immunostaining and expression of tagged protein, and Phc1 unusual in that in many instances it was detected on only one of the two Xis (unpublished data). In cells assayed for localization of macroH2A and Phc1, both proteins exhibited some overlapping sites of enrichment at regions other than the Xi. Similar results were observed when macroH2A was examined in combination with other mPRC1 proteins (unpublished data), suggesting that localization of mPRC1 proteins and this variant histone may be regulated by related mechanisms. These data demonstrate that Bmi-1, Cbx2, and Phc2 exhibit Xi localization in a significant proportion of cells, indicating a role for mPRC1 proteins in X inactivation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA. plath@wi.mit.edu

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.

Show MeSH
Related in: MedlinePlus