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Histone variant macroH2A confers resistance to nuclear reprogramming.

Pasque V, Gillich A, Garrett N, Gurdon JB - EMBO J. (2011)

Bottom Line: Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi.Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs.Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Cambridge, UK. v.pasque@gurdon.cam.ac.uk

ABSTRACT
How various layers of epigenetic repression restrict somatic cell nuclear reprogramming is poorly understood. The transfer of mammalian somatic cell nuclei into Xenopus oocytes induces transcriptional reprogramming of previously repressed genes. Here, we address the mechanisms that restrict reprogramming following nuclear transfer by assessing the stability of the inactive X chromosome (Xi) in different stages of inactivation. We find that the Xi of mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by nuclear transfer, while the Xi of differentiated or extraembryonic cells is irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi. Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.

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The Xi of EpiSCs can be reactivated by nuclear transfer to Xenopus oocytes. (A) Schematic representation of Xi-GFP EpiSCs nuclear transfer experiments. Undifferentiated female EpiSCs cultured on feeders were sorted from differentiating cells by flow cytometry of SSEA1-positive, GFP-negative EpiSCs. After SLO permeabilization, Xi-GFP EpiSC nuclei were transplanted to oocyte GV, and the resulting oocytes were cultured for 3 days. (B) Xi-GFP of EpiSC nuclei can be reactivated after nuclear transfer. Quantitative RT–PCR of X-GFP expression after nuclear transfer. Time points and types of transplanted nuclei are indicated. Transcript levels are shown in table±s.e.m. P<0.05, except samples marked *P<0.2, n=3, error bars show s.e.m. a.u. represents arbitrary unit. (C) Rlim allele-specific RT–PCR. Validation of allele-specific Rlim RT–PCR on cells derived from embryos resulting from a cross between X-GFP Musculus and Castaneus mice (maternal genotype denoted first). MEFs and EpiSCs were derived from embryos genotyped for sex (Ube1 expression) and X-GFP transgene expression and sorted by flow cytometry based on GFP expression. (D) Rlim can be reactivated after nuclear transfer. Allele-specific Rlim RT–PCR of Xi-GFP mus/cast MEFs and EpiSCs, immediately after (day 0) or on day 3 after nuclear transfer.
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f2: The Xi of EpiSCs can be reactivated by nuclear transfer to Xenopus oocytes. (A) Schematic representation of Xi-GFP EpiSCs nuclear transfer experiments. Undifferentiated female EpiSCs cultured on feeders were sorted from differentiating cells by flow cytometry of SSEA1-positive, GFP-negative EpiSCs. After SLO permeabilization, Xi-GFP EpiSC nuclei were transplanted to oocyte GV, and the resulting oocytes were cultured for 3 days. (B) Xi-GFP of EpiSC nuclei can be reactivated after nuclear transfer. Quantitative RT–PCR of X-GFP expression after nuclear transfer. Time points and types of transplanted nuclei are indicated. Transcript levels are shown in table±s.e.m. P<0.05, except samples marked *P<0.2, n=3, error bars show s.e.m. a.u. represents arbitrary unit. (C) Rlim allele-specific RT–PCR. Validation of allele-specific Rlim RT–PCR on cells derived from embryos resulting from a cross between X-GFP Musculus and Castaneus mice (maternal genotype denoted first). MEFs and EpiSCs were derived from embryos genotyped for sex (Ube1 expression) and X-GFP transgene expression and sorted by flow cytometry based on GFP expression. (D) Rlim can be reactivated after nuclear transfer. Allele-specific Rlim RT–PCR of Xi-GFP mus/cast MEFs and EpiSCs, immediately after (day 0) or on day 3 after nuclear transfer.

Mentions: We hypothesized that if resistance to Xi(diff) gene reactivation is under the regulation of epigenetic modifications, the Xi of cells that are less differentiated might carry less repressive marks and be reactivated after nuclear transfer to oocytes. To test this, we used EpiSCs, derived from mouse post-implantation epiblast—the least differentiated cell type known to have undergone XCI (Brons et al, 2007; Tesar et al, 2007). Female EpiSCs have one of their two X chromosomes inactivated, while expressing the autosomal pluripotency genes Oct4, Sox2 and Nanog. The stability of the Xi of EpiSCs is not known so far. We asked if the Xi of EpiSCs (Xi Epi) can be reactivated after nuclear transfer of EpiSC nuclei, again following Xi-GFP expression. We derived X-GFP EpiSCs from E6.5 epiblasts and established Xi-GFP EpiSC lines. We confirmed that female EpiSCs had undergone XCI, and contained an Xi, while expressing pluripotency markers (Supplementary Figures S3 and S4B). To eliminate occasional differentiating EpiSCs or feeder cells from the cultures, we used flow cytometry to separate undifferentiated EpiSCs expressing the pluripotent marker SSEA1 from differentiating, SSEA1-negative cells (Supplementary Figure S5). We transplanted sorted Xi-GFP EpiSCs nuclei to oocyte GVs as depicted in Figure 2A. We also transplanted Xi-GFP and Xa-GFP MEF nuclei (SSEA1 negative) for comparison. While Xi-GFP (diff) of MEF nuclei was not reactivated, the Xi-GFP (Epi) of EpiSC nuclei was strongly reactivated 3 days after nuclear transfer, to a level comparable to that of Xa-GFP MEF nuclei on day 0 (Figure 2B). This indicated that the Xi of EpiSCs is not resistant to reprogramming by oocytes, unlike the Xi of differentiated cells. Similar results were obtained when we transplanted the nuclei of feeder-free EpiSCs cultured on fibronectin (not shown). To test whether endogenous X-linked genes are also reactivated from transplanted EpiSCs nuclei, we carried out allele-specific RT–PCR by exploiting a known polymorphism in X-linked gene Rlim (Huynh and Lee, 2003). We derived Xi-GFP MEFs and EpiSCs from embryos obtained by crossing X-GFP Mus musculus and Mus castaneus mice. Figure 2C shows that restriction enzyme sites present in the musculus, but not the castaneus allele allow to identify the expression origin of the RT–PCR product. We transplanted Xi-GFP MEF and Xi-GFP EpiSC nuclei into oocyte GVs and assayed Rlim expression on day 0 and day 3 after nuclear transfer. Three days after nuclear transfer, monoallelic Rlim expression was detected from transplanted Xi-GFP MEF nuclei, while biallelic expression was detected from transplanted Xi-GFP EpiSCs (Figure 2D). Therefore, Rlim can be reactivated from the Xi after nuclear transfer. These results suggest that the epigenetic inactivation of the Xi in EpiSCs is much less resistant to reprogramming by oocytes than the Xi of differentiated cells.


Histone variant macroH2A confers resistance to nuclear reprogramming.

Pasque V, Gillich A, Garrett N, Gurdon JB - EMBO J. (2011)

The Xi of EpiSCs can be reactivated by nuclear transfer to Xenopus oocytes. (A) Schematic representation of Xi-GFP EpiSCs nuclear transfer experiments. Undifferentiated female EpiSCs cultured on feeders were sorted from differentiating cells by flow cytometry of SSEA1-positive, GFP-negative EpiSCs. After SLO permeabilization, Xi-GFP EpiSC nuclei were transplanted to oocyte GV, and the resulting oocytes were cultured for 3 days. (B) Xi-GFP of EpiSC nuclei can be reactivated after nuclear transfer. Quantitative RT–PCR of X-GFP expression after nuclear transfer. Time points and types of transplanted nuclei are indicated. Transcript levels are shown in table±s.e.m. P<0.05, except samples marked *P<0.2, n=3, error bars show s.e.m. a.u. represents arbitrary unit. (C) Rlim allele-specific RT–PCR. Validation of allele-specific Rlim RT–PCR on cells derived from embryos resulting from a cross between X-GFP Musculus and Castaneus mice (maternal genotype denoted first). MEFs and EpiSCs were derived from embryos genotyped for sex (Ube1 expression) and X-GFP transgene expression and sorted by flow cytometry based on GFP expression. (D) Rlim can be reactivated after nuclear transfer. Allele-specific Rlim RT–PCR of Xi-GFP mus/cast MEFs and EpiSCs, immediately after (day 0) or on day 3 after nuclear transfer.
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f2: The Xi of EpiSCs can be reactivated by nuclear transfer to Xenopus oocytes. (A) Schematic representation of Xi-GFP EpiSCs nuclear transfer experiments. Undifferentiated female EpiSCs cultured on feeders were sorted from differentiating cells by flow cytometry of SSEA1-positive, GFP-negative EpiSCs. After SLO permeabilization, Xi-GFP EpiSC nuclei were transplanted to oocyte GV, and the resulting oocytes were cultured for 3 days. (B) Xi-GFP of EpiSC nuclei can be reactivated after nuclear transfer. Quantitative RT–PCR of X-GFP expression after nuclear transfer. Time points and types of transplanted nuclei are indicated. Transcript levels are shown in table±s.e.m. P<0.05, except samples marked *P<0.2, n=3, error bars show s.e.m. a.u. represents arbitrary unit. (C) Rlim allele-specific RT–PCR. Validation of allele-specific Rlim RT–PCR on cells derived from embryos resulting from a cross between X-GFP Musculus and Castaneus mice (maternal genotype denoted first). MEFs and EpiSCs were derived from embryos genotyped for sex (Ube1 expression) and X-GFP transgene expression and sorted by flow cytometry based on GFP expression. (D) Rlim can be reactivated after nuclear transfer. Allele-specific Rlim RT–PCR of Xi-GFP mus/cast MEFs and EpiSCs, immediately after (day 0) or on day 3 after nuclear transfer.
Mentions: We hypothesized that if resistance to Xi(diff) gene reactivation is under the regulation of epigenetic modifications, the Xi of cells that are less differentiated might carry less repressive marks and be reactivated after nuclear transfer to oocytes. To test this, we used EpiSCs, derived from mouse post-implantation epiblast—the least differentiated cell type known to have undergone XCI (Brons et al, 2007; Tesar et al, 2007). Female EpiSCs have one of their two X chromosomes inactivated, while expressing the autosomal pluripotency genes Oct4, Sox2 and Nanog. The stability of the Xi of EpiSCs is not known so far. We asked if the Xi of EpiSCs (Xi Epi) can be reactivated after nuclear transfer of EpiSC nuclei, again following Xi-GFP expression. We derived X-GFP EpiSCs from E6.5 epiblasts and established Xi-GFP EpiSC lines. We confirmed that female EpiSCs had undergone XCI, and contained an Xi, while expressing pluripotency markers (Supplementary Figures S3 and S4B). To eliminate occasional differentiating EpiSCs or feeder cells from the cultures, we used flow cytometry to separate undifferentiated EpiSCs expressing the pluripotent marker SSEA1 from differentiating, SSEA1-negative cells (Supplementary Figure S5). We transplanted sorted Xi-GFP EpiSCs nuclei to oocyte GVs as depicted in Figure 2A. We also transplanted Xi-GFP and Xa-GFP MEF nuclei (SSEA1 negative) for comparison. While Xi-GFP (diff) of MEF nuclei was not reactivated, the Xi-GFP (Epi) of EpiSC nuclei was strongly reactivated 3 days after nuclear transfer, to a level comparable to that of Xa-GFP MEF nuclei on day 0 (Figure 2B). This indicated that the Xi of EpiSCs is not resistant to reprogramming by oocytes, unlike the Xi of differentiated cells. Similar results were obtained when we transplanted the nuclei of feeder-free EpiSCs cultured on fibronectin (not shown). To test whether endogenous X-linked genes are also reactivated from transplanted EpiSCs nuclei, we carried out allele-specific RT–PCR by exploiting a known polymorphism in X-linked gene Rlim (Huynh and Lee, 2003). We derived Xi-GFP MEFs and EpiSCs from embryos obtained by crossing X-GFP Mus musculus and Mus castaneus mice. Figure 2C shows that restriction enzyme sites present in the musculus, but not the castaneus allele allow to identify the expression origin of the RT–PCR product. We transplanted Xi-GFP MEF and Xi-GFP EpiSC nuclei into oocyte GVs and assayed Rlim expression on day 0 and day 3 after nuclear transfer. Three days after nuclear transfer, monoallelic Rlim expression was detected from transplanted Xi-GFP MEF nuclei, while biallelic expression was detected from transplanted Xi-GFP EpiSCs (Figure 2D). Therefore, Rlim can be reactivated from the Xi after nuclear transfer. These results suggest that the epigenetic inactivation of the Xi in EpiSCs is much less resistant to reprogramming by oocytes than the Xi of differentiated cells.

Bottom Line: Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi.Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs.Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Cambridge, UK. v.pasque@gurdon.cam.ac.uk

ABSTRACT
How various layers of epigenetic repression restrict somatic cell nuclear reprogramming is poorly understood. The transfer of mammalian somatic cell nuclei into Xenopus oocytes induces transcriptional reprogramming of previously repressed genes. Here, we address the mechanisms that restrict reprogramming following nuclear transfer by assessing the stability of the inactive X chromosome (Xi) in different stages of inactivation. We find that the Xi of mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by nuclear transfer, while the Xi of differentiated or extraembryonic cells is irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi. Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.

Show MeSH