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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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mH2A depletion improves reprogramming by nuclear transfer. (A) qRT–PCR analysis of mH2A1 and mH2A2 expression following shRNA-mediated mH2A RNAi. (B) Western analysis of mH2A1 in shRNA expressing Xi-GFP MEFs. (C, D) qPCR analysis of GFP (black), Sox2 (grey) and Oct4 (white) expression in transplanted Xi-GFP MEFs nuclei subjected to mH2A RNAi and/or TSA treatment. P<0.05 except samples marked *P<0.06 in (C), or *P<0.08 in (D), n=3. Error bars are s.e.m. Note the differences in y axis.
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f8: mH2A depletion improves reprogramming by nuclear transfer. (A) qRT–PCR analysis of mH2A1 and mH2A2 expression following shRNA-mediated mH2A RNAi. (B) Western analysis of mH2A1 in shRNA expressing Xi-GFP MEFs. (C, D) qPCR analysis of GFP (black), Sox2 (grey) and Oct4 (white) expression in transplanted Xi-GFP MEFs nuclei subjected to mH2A RNAi and/or TSA treatment. P<0.05 except samples marked *P<0.06 in (C), or *P<0.08 in (D), n=3. Error bars are s.e.m. Note the differences in y axis.

Mentions: To test if incorporation of mH2A into chromatin restricts transcriptional reactivation after nuclear transfer, we established Xi-GFP MEF lines stably expressing shRNAs against mH2A1 (mH2A1.1 and mH2A1.2), macroH2A2 (mH2A2), control scramble sequence or both mH2A1 and mH2A2 (Figure 8A and B). mH2A depletion alone did not induce reactivation of Xi-GFP, Sox2 or Oct4 before nuclear transfer (Supplementary Figure S8A and B), except for a 2.5-fold increase over background in Oct4 transcripts upon co-depletion of mH2A1 and mH2A2. We transplanted the nuclei of mH2A depleted and control Xi-GFP MEFs to oocyte GV and analysed transcriptional reactivation 2 days after nuclear transfer (Figure 8C and D). mH2A knockdown was not sufficient for full reactivation of Xi-GFP, when compared with Xa-GFP transcript levels from transplanted Xa-GFP MEFs. However, mH2A depletion led to a significant, 1.7- to 2.4-fold increase over background in detected GFP transcripts (Figure 8C). This increase was comparable to the increase seen in transplanted oocytes grown in the presence of the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) (two-fold; Figure 8C). Moreover, depletion of mH2A1 and mH2A2 together with TSA treatment resulted in the combined effect of mH2A knockdown and TSA alone namely a 2.8-fold increase in GFP transcripts. We conclude that mH2A is not the only factor contributing to Xi reversibility, yet mH2A does restrict transcriptional reprogramming by oocytes. To address whether mH2A may be a more general restriction to gene reactivation, we analysed transcript levels of pluripotency genes Sox2 and Oct4 after nuclear transfer of mH2A depleted cells. Strikingly, the effect of mH2A depletion was even more pronounced, with a 1.6- to 3.1-fold and a 3.1- to 8.2-fold increase in Sox2 and Oct4 reactivation, respectively (Figure 8D). Sox2 and Oct4 reactivation were increased 3.9- and 7.9-fold by TSA alone, and 7.2- and 15.6-fold by TSA together with mH2A1 and mH2A2 co-depletion. We conclude that mH2A contributes to resistance to transcriptional reprogramming.


Histone variant macroH2A confers resistance to nuclear reprogramming.

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

mH2A depletion improves reprogramming by nuclear transfer. (A) qRT–PCR analysis of mH2A1 and mH2A2 expression following shRNA-mediated mH2A RNAi. (B) Western analysis of mH2A1 in shRNA expressing Xi-GFP MEFs. (C, D) qPCR analysis of GFP (black), Sox2 (grey) and Oct4 (white) expression in transplanted Xi-GFP MEFs nuclei subjected to mH2A RNAi and/or TSA treatment. P<0.05 except samples marked *P<0.06 in (C), or *P<0.08 in (D), n=3. Error bars are s.e.m. Note the differences in y axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f8: mH2A depletion improves reprogramming by nuclear transfer. (A) qRT–PCR analysis of mH2A1 and mH2A2 expression following shRNA-mediated mH2A RNAi. (B) Western analysis of mH2A1 in shRNA expressing Xi-GFP MEFs. (C, D) qPCR analysis of GFP (black), Sox2 (grey) and Oct4 (white) expression in transplanted Xi-GFP MEFs nuclei subjected to mH2A RNAi and/or TSA treatment. P<0.05 except samples marked *P<0.06 in (C), or *P<0.08 in (D), n=3. Error bars are s.e.m. Note the differences in y axis.
Mentions: To test if incorporation of mH2A into chromatin restricts transcriptional reactivation after nuclear transfer, we established Xi-GFP MEF lines stably expressing shRNAs against mH2A1 (mH2A1.1 and mH2A1.2), macroH2A2 (mH2A2), control scramble sequence or both mH2A1 and mH2A2 (Figure 8A and B). mH2A depletion alone did not induce reactivation of Xi-GFP, Sox2 or Oct4 before nuclear transfer (Supplementary Figure S8A and B), except for a 2.5-fold increase over background in Oct4 transcripts upon co-depletion of mH2A1 and mH2A2. We transplanted the nuclei of mH2A depleted and control Xi-GFP MEFs to oocyte GV and analysed transcriptional reactivation 2 days after nuclear transfer (Figure 8C and D). mH2A knockdown was not sufficient for full reactivation of Xi-GFP, when compared with Xa-GFP transcript levels from transplanted Xa-GFP MEFs. However, mH2A depletion led to a significant, 1.7- to 2.4-fold increase over background in detected GFP transcripts (Figure 8C). This increase was comparable to the increase seen in transplanted oocytes grown in the presence of the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) (two-fold; Figure 8C). Moreover, depletion of mH2A1 and mH2A2 together with TSA treatment resulted in the combined effect of mH2A knockdown and TSA alone namely a 2.8-fold increase in GFP transcripts. We conclude that mH2A is not the only factor contributing to Xi reversibility, yet mH2A does restrict transcriptional reprogramming by oocytes. To address whether mH2A may be a more general restriction to gene reactivation, we analysed transcript levels of pluripotency genes Sox2 and Oct4 after nuclear transfer of mH2A depleted cells. Strikingly, the effect of mH2A depletion was even more pronounced, with a 1.6- to 3.1-fold and a 3.1- to 8.2-fold increase in Sox2 and Oct4 reactivation, respectively (Figure 8D). Sox2 and Oct4 reactivation were increased 3.9- and 7.9-fold by TSA alone, and 7.2- and 15.6-fold by TSA together with mH2A1 and mH2A2 co-depletion. We conclude that mH2A contributes to resistance to transcriptional reprogramming.

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