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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 long noncoding RNA Xist dissociates from chromatin of the Xi after nuclear transfer. (A) RNA FISH for Xist RNA (green) on transplanted female MEF nuclei. Oocyte GVs containing transplanted nuclei were dissected, fixed and subjected to RNA FISH against Xist RNA. Confocal images reveal that the Xist RNA cloud of female MEFs (0 h) is lost from the Xi 18 h after nuclear transfer. Note the presence of punctate Xist RNA FISH signal dispersed throughout the nucleus of some of the 18 h transplanted nuclei. DAPI is shown in magenta. Low (scale bars=25 μm) and high (scale bars=5 μm) magnification pictures are shown. P denotes permeabilized nuclei. Images are projected Z-sections. (B, C) Xist RNA is lost from the Xi after nuclear transfer of female MEFs (B) and female EpiSCs (C). Xist RNA FISH of nuclear transfer female MEFs and EpiSCs. Samples were collected and fixed at indicated time points. The Xist RNA cloud characteristic of the Xi is maintained up to 3 h after nuclear transfer, then decreases to give a pinpoint signal at 12 and 16 h, and is completely lost from transplanted nuclei by 24–48 h after nuclear transfer. The proportion of nuclei with a Xist RNA cloud is indicated. DAPI is shown in magenta. n=number of nuclei. Scale bars=5 μm in (B) and 2 μm in (C). Images are projected Z-sections. (D) Xist expression levels in transplanted female MEF and EpiSC nuclei. qRT–PCR analysis of Xist (dark grey) and Sox2 (light grey) expression in transplanted nuclei. Xist transcript levels increase after nuclear transfer. Error bars are s.e.m. a.u. represents arbitrary unit.
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f5: The long noncoding RNA Xist dissociates from chromatin of the Xi after nuclear transfer. (A) RNA FISH for Xist RNA (green) on transplanted female MEF nuclei. Oocyte GVs containing transplanted nuclei were dissected, fixed and subjected to RNA FISH against Xist RNA. Confocal images reveal that the Xist RNA cloud of female MEFs (0 h) is lost from the Xi 18 h after nuclear transfer. Note the presence of punctate Xist RNA FISH signal dispersed throughout the nucleus of some of the 18 h transplanted nuclei. DAPI is shown in magenta. Low (scale bars=25 μm) and high (scale bars=5 μm) magnification pictures are shown. P denotes permeabilized nuclei. Images are projected Z-sections. (B, C) Xist RNA is lost from the Xi after nuclear transfer of female MEFs (B) and female EpiSCs (C). Xist RNA FISH of nuclear transfer female MEFs and EpiSCs. Samples were collected and fixed at indicated time points. The Xist RNA cloud characteristic of the Xi is maintained up to 3 h after nuclear transfer, then decreases to give a pinpoint signal at 12 and 16 h, and is completely lost from transplanted nuclei by 24–48 h after nuclear transfer. The proportion of nuclei with a Xist RNA cloud is indicated. DAPI is shown in magenta. n=number of nuclei. Scale bars=5 μm in (B) and 2 μm in (C). Images are projected Z-sections. (D) Xist expression levels in transplanted female MEF and EpiSC nuclei. qRT–PCR analysis of Xist (dark grey) and Sox2 (light grey) expression in transplanted nuclei. Xist transcript levels increase after nuclear transfer. Error bars are s.e.m. a.u. represents arbitrary unit.

Mentions: During initiation of XCI, the long noncoding RNA Xist induces gene inactivation on the chromosome from which it is produced, by recruiting the machinery necessary for silencing (Heard and Disteche, 2006). Because X reactivation is associated with the removal of Xist RNA, we investigated Xist RNA localization on the Xi in nuclei of female somatic cells transplanted into oocytes. Moreover, the fate of long noncoding RNAs has not previously been described following somatic cell nuclear transfer to Xenopus oocytes. We followed the localization of Xist RNA before and after nuclear transfer by fluorescent RNA in situ hybridization (RNA FISH). RNA FISH against Xist identified a single Xist RNA cloud localized to the Xi of untransplanted female MEFs and EpiSCs (Supplementary Figure S4A and B, respectively). Strikingly, Xist RNA coating of the Xi was lost in transplanted MEF nuclei 18 h after nuclear transfer, although it was fully localized to the Xi immediately after transfer (Figure 5A). A detailed time course revealed that nuclear transfer did not induce obvious changes to this pattern within 3 h after transfer (Figure 5B). However after this, Xist RNA was gradually lost, and was fully delocalized from the Xi after 12 h. Whereas over 80% of transplanted MEF nuclei contained an Xist RNA cloud on their Xi within 3 h after transfer (n=44–163), none of the transplanted MEF nuclei had Xist RNA on their Xi 12 h (3%) and 24 h (0%) after nuclear transfer (n=90 and 158; Figure 4B). In some instances, Xist RNA dispersion was seen, with multiple Xist RNA FISH punctate signals distributed throughout transplanted nuclei, reminiscent of those observed in mitotic cells (Figure 5A (18 h) and Supplementary Figure S5B, high magnification panels). Loss of Xist RNA from the Xi was also observed with similar kinetics in the nuclei of transplanted female EpiSCs (Figure 5C), with near complete loss of the Xist RNA cloud from the Xi 24 h after nuclear transfer (Figure 5C). Visualization of the Xi chromosome territory by H3K27me3 immunofluorescence revealed no obvious change in the shape of the Xi, suggesting that Xist delocalization is not due to changes in Xi organization (Figure 4B). Together, these results show that the long noncoding RNA Xist is dispersed from the Xi domain of both Xi(diff) and Xi(Epi) after somatic cell nuclear transfer to oocyte GVs.


Histone variant macroH2A confers resistance to nuclear reprogramming.

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

The long noncoding RNA Xist dissociates from chromatin of the Xi after nuclear transfer. (A) RNA FISH for Xist RNA (green) on transplanted female MEF nuclei. Oocyte GVs containing transplanted nuclei were dissected, fixed and subjected to RNA FISH against Xist RNA. Confocal images reveal that the Xist RNA cloud of female MEFs (0 h) is lost from the Xi 18 h after nuclear transfer. Note the presence of punctate Xist RNA FISH signal dispersed throughout the nucleus of some of the 18 h transplanted nuclei. DAPI is shown in magenta. Low (scale bars=25 μm) and high (scale bars=5 μm) magnification pictures are shown. P denotes permeabilized nuclei. Images are projected Z-sections. (B, C) Xist RNA is lost from the Xi after nuclear transfer of female MEFs (B) and female EpiSCs (C). Xist RNA FISH of nuclear transfer female MEFs and EpiSCs. Samples were collected and fixed at indicated time points. The Xist RNA cloud characteristic of the Xi is maintained up to 3 h after nuclear transfer, then decreases to give a pinpoint signal at 12 and 16 h, and is completely lost from transplanted nuclei by 24–48 h after nuclear transfer. The proportion of nuclei with a Xist RNA cloud is indicated. DAPI is shown in magenta. n=number of nuclei. Scale bars=5 μm in (B) and 2 μm in (C). Images are projected Z-sections. (D) Xist expression levels in transplanted female MEF and EpiSC nuclei. qRT–PCR analysis of Xist (dark grey) and Sox2 (light grey) expression in transplanted nuclei. Xist transcript levels increase after nuclear transfer. Error bars are s.e.m. a.u. represents arbitrary unit.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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f5: The long noncoding RNA Xist dissociates from chromatin of the Xi after nuclear transfer. (A) RNA FISH for Xist RNA (green) on transplanted female MEF nuclei. Oocyte GVs containing transplanted nuclei were dissected, fixed and subjected to RNA FISH against Xist RNA. Confocal images reveal that the Xist RNA cloud of female MEFs (0 h) is lost from the Xi 18 h after nuclear transfer. Note the presence of punctate Xist RNA FISH signal dispersed throughout the nucleus of some of the 18 h transplanted nuclei. DAPI is shown in magenta. Low (scale bars=25 μm) and high (scale bars=5 μm) magnification pictures are shown. P denotes permeabilized nuclei. Images are projected Z-sections. (B, C) Xist RNA is lost from the Xi after nuclear transfer of female MEFs (B) and female EpiSCs (C). Xist RNA FISH of nuclear transfer female MEFs and EpiSCs. Samples were collected and fixed at indicated time points. The Xist RNA cloud characteristic of the Xi is maintained up to 3 h after nuclear transfer, then decreases to give a pinpoint signal at 12 and 16 h, and is completely lost from transplanted nuclei by 24–48 h after nuclear transfer. The proportion of nuclei with a Xist RNA cloud is indicated. DAPI is shown in magenta. n=number of nuclei. Scale bars=5 μm in (B) and 2 μm in (C). Images are projected Z-sections. (D) Xist expression levels in transplanted female MEF and EpiSC nuclei. qRT–PCR analysis of Xist (dark grey) and Sox2 (light grey) expression in transplanted nuclei. Xist transcript levels increase after nuclear transfer. Error bars are s.e.m. a.u. represents arbitrary unit.
Mentions: During initiation of XCI, the long noncoding RNA Xist induces gene inactivation on the chromosome from which it is produced, by recruiting the machinery necessary for silencing (Heard and Disteche, 2006). Because X reactivation is associated with the removal of Xist RNA, we investigated Xist RNA localization on the Xi in nuclei of female somatic cells transplanted into oocytes. Moreover, the fate of long noncoding RNAs has not previously been described following somatic cell nuclear transfer to Xenopus oocytes. We followed the localization of Xist RNA before and after nuclear transfer by fluorescent RNA in situ hybridization (RNA FISH). RNA FISH against Xist identified a single Xist RNA cloud localized to the Xi of untransplanted female MEFs and EpiSCs (Supplementary Figure S4A and B, respectively). Strikingly, Xist RNA coating of the Xi was lost in transplanted MEF nuclei 18 h after nuclear transfer, although it was fully localized to the Xi immediately after transfer (Figure 5A). A detailed time course revealed that nuclear transfer did not induce obvious changes to this pattern within 3 h after transfer (Figure 5B). However after this, Xist RNA was gradually lost, and was fully delocalized from the Xi after 12 h. Whereas over 80% of transplanted MEF nuclei contained an Xist RNA cloud on their Xi within 3 h after transfer (n=44–163), none of the transplanted MEF nuclei had Xist RNA on their Xi 12 h (3%) and 24 h (0%) after nuclear transfer (n=90 and 158; Figure 4B). In some instances, Xist RNA dispersion was seen, with multiple Xist RNA FISH punctate signals distributed throughout transplanted nuclei, reminiscent of those observed in mitotic cells (Figure 5A (18 h) and Supplementary Figure S5B, high magnification panels). Loss of Xist RNA from the Xi was also observed with similar kinetics in the nuclei of transplanted female EpiSCs (Figure 5C), with near complete loss of the Xist RNA cloud from the Xi 24 h after nuclear transfer (Figure 5C). Visualization of the Xi chromosome territory by H3K27me3 immunofluorescence revealed no obvious change in the shape of the Xi, suggesting that Xist delocalization is not due to changes in Xi organization (Figure 4B). Together, these results show that the long noncoding RNA Xist is dispersed from the Xi domain of both Xi(diff) and Xi(Epi) after somatic cell nuclear transfer to oocyte GVs.

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