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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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Nuclear transfer reverses epigenetically stable, Xist-induced and Xist-independent gene repression. Reversibility of PGK-puro silencing following nuclear transfer of clone 36 cells. To obtain the Xist-dependent (XD) PGK-puro repressed state, clone 36 ES cells were induced to express Xist for 4 days. To obtain the Xist-independent (XI), stable PGK-puro repressed state, clone 36 ES cells were induced to differentiate with RA for 4 days while being induced with Xist at the same time. The nuclei of XD and XI PGK-puro repressed cells were transplanted to oocytes. Biological triplicates were collected immediately or 2 days after nuclear transfer. Nuclei induced to ectopically express Xist after nuclear transfer, within the GV is indicated (+). Transcriptional analysis of puro (dark grey) and Xist (light grey) expression by qRT–PCR of oocytes transplanted with nuclei obtained as described in Supplementary Figure S6B is shown. P<0.05, n=3. Error bars are s.e.m. a.u. represents arbitrary unit.
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f6: Nuclear transfer reverses epigenetically stable, Xist-induced and Xist-independent gene repression. Reversibility of PGK-puro silencing following nuclear transfer of clone 36 cells. To obtain the Xist-dependent (XD) PGK-puro repressed state, clone 36 ES cells were induced to express Xist for 4 days. To obtain the Xist-independent (XI), stable PGK-puro repressed state, clone 36 ES cells were induced to differentiate with RA for 4 days while being induced with Xist at the same time. The nuclei of XD and XI PGK-puro repressed cells were transplanted to oocytes. Biological triplicates were collected immediately or 2 days after nuclear transfer. Nuclei induced to ectopically express Xist after nuclear transfer, within the GV is indicated (+). Transcriptional analysis of puro (dark grey) and Xist (light grey) expression by qRT–PCR of oocytes transplanted with nuclei obtained as described in Supplementary Figure S6B is shown. P<0.05, n=3. Error bars are s.e.m. a.u. represents arbitrary unit.

Mentions: By using an independent system, allowing controlled Xist expression, we tested whether nuclear transfer to oocyte reactivates genes that are maintained in a repressed state in a Xist RNA-dependent or -independent manner. An inducible Xist expression system in ES cells triggers silencing of a PGK-puromycin reporter (PGK-puro) in cis (Wutz and Jaenisch, 2000). This system has been shown to induce reversible, XD PGK-puro repression in ES cells or stable silencing upon combined Xist induction and retinoic acid (RA) ES cells differentiation (Supplementary Figure S6; Wutz and Jaenisch, 2000; Leeb and Wutz, 2007). This is based on PGK-puro reactivation upon removal of Xist after a period during which repression has been triggered by Xist. We tested reactivation of repressed PGK-puro from XD or stable XI cells, by nuclear transfer to Xenopus oocytes. Expression analysis showed a strong reactivation of PGK-puro expression from both types of transplanted nuclei (Figure 6, lanes 1–4). This means that the epigenetically stable repression of PGK-puro induced by Xist during RA differentiation of ES cells is efficiently reprogrammed by Xenopus oocytes. Therefore, we reasoned that the resistance of the Xi of MEFs to reactivation by Xenopus oocytes must be acquired late in the XCI process, even after epigenetically stable, XI repression is induced. This prompted us to examine the incorporation of the repressive histone variant mH2A, a known late event of XCI (see below).


Histone variant macroH2A confers resistance to nuclear reprogramming.

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

Nuclear transfer reverses epigenetically stable, Xist-induced and Xist-independent gene repression. Reversibility of PGK-puro silencing following nuclear transfer of clone 36 cells. To obtain the Xist-dependent (XD) PGK-puro repressed state, clone 36 ES cells were induced to express Xist for 4 days. To obtain the Xist-independent (XI), stable PGK-puro repressed state, clone 36 ES cells were induced to differentiate with RA for 4 days while being induced with Xist at the same time. The nuclei of XD and XI PGK-puro repressed cells were transplanted to oocytes. Biological triplicates were collected immediately or 2 days after nuclear transfer. Nuclei induced to ectopically express Xist after nuclear transfer, within the GV is indicated (+). Transcriptional analysis of puro (dark grey) and Xist (light grey) expression by qRT–PCR of oocytes transplanted with nuclei obtained as described in Supplementary Figure S6B is shown. P<0.05, n=3. Error bars are s.e.m. a.u. represents arbitrary unit.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116279&req=5

f6: Nuclear transfer reverses epigenetically stable, Xist-induced and Xist-independent gene repression. Reversibility of PGK-puro silencing following nuclear transfer of clone 36 cells. To obtain the Xist-dependent (XD) PGK-puro repressed state, clone 36 ES cells were induced to express Xist for 4 days. To obtain the Xist-independent (XI), stable PGK-puro repressed state, clone 36 ES cells were induced to differentiate with RA for 4 days while being induced with Xist at the same time. The nuclei of XD and XI PGK-puro repressed cells were transplanted to oocytes. Biological triplicates were collected immediately or 2 days after nuclear transfer. Nuclei induced to ectopically express Xist after nuclear transfer, within the GV is indicated (+). Transcriptional analysis of puro (dark grey) and Xist (light grey) expression by qRT–PCR of oocytes transplanted with nuclei obtained as described in Supplementary Figure S6B is shown. P<0.05, n=3. Error bars are s.e.m. a.u. represents arbitrary unit.
Mentions: By using an independent system, allowing controlled Xist expression, we tested whether nuclear transfer to oocyte reactivates genes that are maintained in a repressed state in a Xist RNA-dependent or -independent manner. An inducible Xist expression system in ES cells triggers silencing of a PGK-puromycin reporter (PGK-puro) in cis (Wutz and Jaenisch, 2000). This system has been shown to induce reversible, XD PGK-puro repression in ES cells or stable silencing upon combined Xist induction and retinoic acid (RA) ES cells differentiation (Supplementary Figure S6; Wutz and Jaenisch, 2000; Leeb and Wutz, 2007). This is based on PGK-puro reactivation upon removal of Xist after a period during which repression has been triggered by Xist. We tested reactivation of repressed PGK-puro from XD or stable XI cells, by nuclear transfer to Xenopus oocytes. Expression analysis showed a strong reactivation of PGK-puro expression from both types of transplanted nuclei (Figure 6, lanes 1–4). This means that the epigenetically stable repression of PGK-puro induced by Xist during RA differentiation of ES cells is efficiently reprogrammed by Xenopus oocytes. Therefore, we reasoned that the resistance of the Xi of MEFs to reactivation by Xenopus oocytes must be acquired late in the XCI process, even after epigenetically stable, XI repression is induced. This prompted us to examine the incorporation of the repressive histone variant mH2A, a known late event of XCI (see below).

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