Limits...
Histone variant macroH2A marks embryonic differentiation in vivo and acts as an epigenetic barrier to induced pluripotency.

Pasque V, Radzisheuskaya A, Gillich A, Halley-Stott RP, Panamarova M, Zernicka-Goetz M, Surani MA, Silva JC - J. Cell. Sci. (2012)

Bottom Line: The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras.In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency.In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, UK. vpasque@cantab.net

ABSTRACT
How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.

Show MeSH

Related in: MedlinePlus

MacroH2A.1 becomes highly expressed during somatic lineage development. (A) E6.5 female X-GFP mouse conceptus wholemount immunofluorescence against macroH2A.1 (red in merge panel) and GFP (green in merge panel). MacroH2A.1 is highly expressed in the visceral endoderm (VE) and to some extent in the extra embryonic ectoderm (TE) but is not detected in the epiblast (EPI), precursor of all somatic lineages (mosaic X-GFP expression due to random X chromosome inactivation). DAPI is in blue. Images are projected confocal Z-sections. (B) Western blot analysis of macroH2A.1 in ESCs, EpiSCs, NSCs and MEFs. All cells are female. Tubulin was used as a loading control. (C) Quantification of western blot analysis shown in B. MacroH2A.1 signal was normalized to tubulin and ESCs levels set to 1. MacroH2A.1 expression is lowest in ESCs and increases with the differentiated state in EpiSCs, MEFs and adult NSCs. (D) E9.5 female X-GFP mouse embryo wholemount immunofluorescence against macroH2A.1 (red) and GFP (green). MacroH2A.1 is expressed throughout all tissues of the embryo at this stage. DAPI is in blue. Images acquired using epifluorescence microscopy. (E) E9.5 X-GFP mouse embryo immunofluorescence against macroH2A.1 (red) and GFP (green) showing a portion of the lateral plate mesoderm (including somites). Nuclear macroH2A.1 is detected in all somatic cells. Mosaic X-GFP expression is due to random X chromosome inactivation. DAPI is in blue. Images are projected confocal Z-sections. Scale bars: 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585521&req=5

f02: MacroH2A.1 becomes highly expressed during somatic lineage development. (A) E6.5 female X-GFP mouse conceptus wholemount immunofluorescence against macroH2A.1 (red in merge panel) and GFP (green in merge panel). MacroH2A.1 is highly expressed in the visceral endoderm (VE) and to some extent in the extra embryonic ectoderm (TE) but is not detected in the epiblast (EPI), precursor of all somatic lineages (mosaic X-GFP expression due to random X chromosome inactivation). DAPI is in blue. Images are projected confocal Z-sections. (B) Western blot analysis of macroH2A.1 in ESCs, EpiSCs, NSCs and MEFs. All cells are female. Tubulin was used as a loading control. (C) Quantification of western blot analysis shown in B. MacroH2A.1 signal was normalized to tubulin and ESCs levels set to 1. MacroH2A.1 expression is lowest in ESCs and increases with the differentiated state in EpiSCs, MEFs and adult NSCs. (D) E9.5 female X-GFP mouse embryo wholemount immunofluorescence against macroH2A.1 (red) and GFP (green). MacroH2A.1 is expressed throughout all tissues of the embryo at this stage. DAPI is in blue. Images acquired using epifluorescence microscopy. (E) E9.5 X-GFP mouse embryo immunofluorescence against macroH2A.1 (red) and GFP (green) showing a portion of the lateral plate mesoderm (including somites). Nuclear macroH2A.1 is detected in all somatic cells. Mosaic X-GFP expression is due to random X chromosome inactivation. DAPI is in blue. Images are projected confocal Z-sections. Scale bars: 20 µm.

Mentions: Next, we carried out wholemount immunofluorescence of E6.5 X-GFP female mouse conceptus against macroH2A.1 and GFP. The paternally derived X-GFP transgene was used as a marker to specifically delineate cells of the epiblast, whose mosaic expression reflects random X chromosome inactivation in female embryos (Hadjantonakis et al., 2001). Remarkably, macroH2A.1 expression was undetectable in the pluripotent epiblast, but it was detected strongly in the nuclei of visceral endoderm cells and to a lesser extent in the nuclei of trophectoderm cells (Fig. 2A; supplementary material Fig. S1A). This mirrors the expression level of macroH2A in cultured cell lines derived from these embryonic lineages, namely epiblast stem cells (EpiSCs) and trophoblast stem (TS) cells (Pasque et al., 2011a).


Histone variant macroH2A marks embryonic differentiation in vivo and acts as an epigenetic barrier to induced pluripotency.

Pasque V, Radzisheuskaya A, Gillich A, Halley-Stott RP, Panamarova M, Zernicka-Goetz M, Surani MA, Silva JC - J. Cell. Sci. (2012)

MacroH2A.1 becomes highly expressed during somatic lineage development. (A) E6.5 female X-GFP mouse conceptus wholemount immunofluorescence against macroH2A.1 (red in merge panel) and GFP (green in merge panel). MacroH2A.1 is highly expressed in the visceral endoderm (VE) and to some extent in the extra embryonic ectoderm (TE) but is not detected in the epiblast (EPI), precursor of all somatic lineages (mosaic X-GFP expression due to random X chromosome inactivation). DAPI is in blue. Images are projected confocal Z-sections. (B) Western blot analysis of macroH2A.1 in ESCs, EpiSCs, NSCs and MEFs. All cells are female. Tubulin was used as a loading control. (C) Quantification of western blot analysis shown in B. MacroH2A.1 signal was normalized to tubulin and ESCs levels set to 1. MacroH2A.1 expression is lowest in ESCs and increases with the differentiated state in EpiSCs, MEFs and adult NSCs. (D) E9.5 female X-GFP mouse embryo wholemount immunofluorescence against macroH2A.1 (red) and GFP (green). MacroH2A.1 is expressed throughout all tissues of the embryo at this stage. DAPI is in blue. Images acquired using epifluorescence microscopy. (E) E9.5 X-GFP mouse embryo immunofluorescence against macroH2A.1 (red) and GFP (green) showing a portion of the lateral plate mesoderm (including somites). Nuclear macroH2A.1 is detected in all somatic cells. Mosaic X-GFP expression is due to random X chromosome inactivation. DAPI is in blue. Images are projected confocal Z-sections. Scale bars: 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585521&req=5

f02: MacroH2A.1 becomes highly expressed during somatic lineage development. (A) E6.5 female X-GFP mouse conceptus wholemount immunofluorescence against macroH2A.1 (red in merge panel) and GFP (green in merge panel). MacroH2A.1 is highly expressed in the visceral endoderm (VE) and to some extent in the extra embryonic ectoderm (TE) but is not detected in the epiblast (EPI), precursor of all somatic lineages (mosaic X-GFP expression due to random X chromosome inactivation). DAPI is in blue. Images are projected confocal Z-sections. (B) Western blot analysis of macroH2A.1 in ESCs, EpiSCs, NSCs and MEFs. All cells are female. Tubulin was used as a loading control. (C) Quantification of western blot analysis shown in B. MacroH2A.1 signal was normalized to tubulin and ESCs levels set to 1. MacroH2A.1 expression is lowest in ESCs and increases with the differentiated state in EpiSCs, MEFs and adult NSCs. (D) E9.5 female X-GFP mouse embryo wholemount immunofluorescence against macroH2A.1 (red) and GFP (green). MacroH2A.1 is expressed throughout all tissues of the embryo at this stage. DAPI is in blue. Images acquired using epifluorescence microscopy. (E) E9.5 X-GFP mouse embryo immunofluorescence against macroH2A.1 (red) and GFP (green) showing a portion of the lateral plate mesoderm (including somites). Nuclear macroH2A.1 is detected in all somatic cells. Mosaic X-GFP expression is due to random X chromosome inactivation. DAPI is in blue. Images are projected confocal Z-sections. Scale bars: 20 µm.
Mentions: Next, we carried out wholemount immunofluorescence of E6.5 X-GFP female mouse conceptus against macroH2A.1 and GFP. The paternally derived X-GFP transgene was used as a marker to specifically delineate cells of the epiblast, whose mosaic expression reflects random X chromosome inactivation in female embryos (Hadjantonakis et al., 2001). Remarkably, macroH2A.1 expression was undetectable in the pluripotent epiblast, but it was detected strongly in the nuclei of visceral endoderm cells and to a lesser extent in the nuclei of trophectoderm cells (Fig. 2A; supplementary material Fig. S1A). This mirrors the expression level of macroH2A in cultured cell lines derived from these embryonic lineages, namely epiblast stem cells (EpiSCs) and trophoblast stem (TS) cells (Pasque et al., 2011a).

Bottom Line: The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras.In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency.In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, UK. vpasque@cantab.net

ABSTRACT
How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert to another fate. The histone variant macroH2A acts as a component of an epigenetic multilayer that heritably maintains the silent X chromosome and has been shown to restrict tumor development. Here we show that macroH2A marks the differentiated cell state during mouse embryogenesis. MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency of induced pluripotency up to 25-fold. The obtained induced pluripotent stem cells reactivated pluripotency genes, silenced retroviral transgenes and contributed to chimeras. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. In summary, our study identifies for the first time a link between an epigenetic mark and cell fate restriction during somatic cell differentiation, which helps to maintain cell identity and antagonizes induction of a pluripotent stem cell state.

Show MeSH
Related in: MedlinePlus