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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 cells lacking Xist 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.
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fig4: Localization of mPRC1 proteins in cells lacking Xist 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.

Mentions: mPRC2-mediated enrichment of H3-3mK27 on the Xi requires continued coating of the Xi by Xist RNA (Plath et al., 2005). Here, we tested whether the Xi localization of mPRC1 proteins also depends on Xist RNA, using female MEFs in which the Xist gene on the Xi is flanked by loxP sites. To delete Xist, cells were infected with adenovirus encoding Cre recombinase. Immunostaining for H3-3mK27 combined with FISH for Xist RNA confirmed that Xist RNA and the Xist-dependent Xi enrichment of H3-3mK27 were lost upon Xist deletion (Fig. 4, a and b). MEFs carrying the conditional Xist allele exhibited enrichment of Bmi-1, Cbx2, and Phc2 on the Xist RNA-coated Xi in an average of 11.2%, 17.3%, and 3.5% of cells respectively (Fig. 4 c). Upon deletion of Xist, an Xi-like focal accumulation of these proteins was no longer detected (Fig. 4 c). Thus, the Xi localization of the mPRC1 proteins Bmi-1, Cbx2, and Phc2, like the enrichment of H3-3mK27, depends on Xist RNA in somatic cells.


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 cells lacking Xist 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.
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Related In: Results  -  Collection

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fig4: Localization of mPRC1 proteins in cells lacking Xist 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.
Mentions: mPRC2-mediated enrichment of H3-3mK27 on the Xi requires continued coating of the Xi by Xist RNA (Plath et al., 2005). Here, we tested whether the Xi localization of mPRC1 proteins also depends on Xist RNA, using female MEFs in which the Xist gene on the Xi is flanked by loxP sites. To delete Xist, cells were infected with adenovirus encoding Cre recombinase. Immunostaining for H3-3mK27 combined with FISH for Xist RNA confirmed that Xist RNA and the Xist-dependent Xi enrichment of H3-3mK27 were lost upon Xist deletion (Fig. 4, a and b). MEFs carrying the conditional Xist allele exhibited enrichment of Bmi-1, Cbx2, and Phc2 on the Xist RNA-coated Xi in an average of 11.2%, 17.3%, and 3.5% of cells respectively (Fig. 4 c). Upon deletion of Xist, an Xi-like focal accumulation of these proteins was no longer detected (Fig. 4 c). Thus, the Xi localization of the mPRC1 proteins Bmi-1, Cbx2, and Phc2, like the enrichment of H3-3mK27, depends on Xist RNA in somatic cells.

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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