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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.

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MacroH2A.1 marks chromatin of regulatory sequences of repressed pluripotency genes in differentiated cells. MacroH2A.1 ChIP analysis of pluripotent and lineage-specific gene regulatory regions in pluripotent (ESCs, light gray), and somatic cells (NSCs, gray; MEFs, dark gray). DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; RR1, regulatory region 1; RR2, regulatory region 2. Error bars depict the s.e.m. (n = 3). There were significant differences between ESCs and NSCs or MEFs, as indicated; *P<0.05; t-test one tail, type 3.
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f03: MacroH2A.1 marks chromatin of regulatory sequences of repressed pluripotency genes in differentiated cells. MacroH2A.1 ChIP analysis of pluripotent and lineage-specific gene regulatory regions in pluripotent (ESCs, light gray), and somatic cells (NSCs, gray; MEFs, dark gray). DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; RR1, regulatory region 1; RR2, regulatory region 2. Error bars depict the s.e.m. (n = 3). There were significant differences between ESCs and NSCs or MEFs, as indicated; *P<0.05; t-test one tail, type 3.

Mentions: Genome-wide analysis of macroH2A variants established that they generally localize to heterochromatic regions (Barzily-Rokni et al., 2011; Buschbeck et al., 2009; Changolkar et al., 2010; Creppe et al., 2012a; Gamble et al., 2010). At the same time, they can also be found in the coding regions of a small proportion of genes whose expression level inversely correlates with the amount of macroH2A incorporated in their chromatin (Gamble and Kraus, 2010; Gamble et al., 2010). Since the presence of macroH2A is mostly considered to have a repressive effect on genes and its abundance correlates with cell differentiation, we hypothesized that it may be incorporated in the chromatin of repressed pluripotency genes in somatic cells. We carried out ChIP analysis in undifferentiated ESCs, MEFs and adult NSCs. In ESCs, macroH2A.1 was detected at low levels in the regulatory sequences of pluripotency genes, Oct4 and Sox2, lineage-specific genes, B-Globin and Thy1, and the housekeeping genes GAPDH and c-Jun (Fig. 3). In somatic cells, both in mouse embryonic fibroblasts (MEFs) and in NSCs, macroH2A.1 became markedly enriched in the regulatory sequences of Oct4, B-Globin and Thy1, correlating with the repressed state of these genes in these cells. MacroH2A.1 did not become enriched in the GAPDH and c-Jun promoter, active in all cell types analyzed (Fig. 3). MacroH2A.1 was highly enriched in the chromatin of Sox2 regulatory regions in MEFs but not in NSCs in which this gene is highly expressed. In conclusion, the histone variant macroH2A.1 incorporates into the nucleosomes in the regulatory sequences of both repressed pluripotency and lineage-specific genes in somatic cells linking macroH2A not only to gene silencing but also to the stabilization of the differentiated cell state.


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 marks chromatin of regulatory sequences of repressed pluripotency genes in differentiated cells. MacroH2A.1 ChIP analysis of pluripotent and lineage-specific gene regulatory regions in pluripotent (ESCs, light gray), and somatic cells (NSCs, gray; MEFs, dark gray). DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; RR1, regulatory region 1; RR2, regulatory region 2. Error bars depict the s.e.m. (n = 3). There were significant differences between ESCs and NSCs or MEFs, as indicated; *P<0.05; t-test one tail, type 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f03: MacroH2A.1 marks chromatin of regulatory sequences of repressed pluripotency genes in differentiated cells. MacroH2A.1 ChIP analysis of pluripotent and lineage-specific gene regulatory regions in pluripotent (ESCs, light gray), and somatic cells (NSCs, gray; MEFs, dark gray). DE, distal enhancer; PE, proximal enhancer; PP, proximal promoter; RR1, regulatory region 1; RR2, regulatory region 2. Error bars depict the s.e.m. (n = 3). There were significant differences between ESCs and NSCs or MEFs, as indicated; *P<0.05; t-test one tail, type 3.
Mentions: Genome-wide analysis of macroH2A variants established that they generally localize to heterochromatic regions (Barzily-Rokni et al., 2011; Buschbeck et al., 2009; Changolkar et al., 2010; Creppe et al., 2012a; Gamble et al., 2010). At the same time, they can also be found in the coding regions of a small proportion of genes whose expression level inversely correlates with the amount of macroH2A incorporated in their chromatin (Gamble and Kraus, 2010; Gamble et al., 2010). Since the presence of macroH2A is mostly considered to have a repressive effect on genes and its abundance correlates with cell differentiation, we hypothesized that it may be incorporated in the chromatin of repressed pluripotency genes in somatic cells. We carried out ChIP analysis in undifferentiated ESCs, MEFs and adult NSCs. In ESCs, macroH2A.1 was detected at low levels in the regulatory sequences of pluripotency genes, Oct4 and Sox2, lineage-specific genes, B-Globin and Thy1, and the housekeeping genes GAPDH and c-Jun (Fig. 3). In somatic cells, both in mouse embryonic fibroblasts (MEFs) and in NSCs, macroH2A.1 became markedly enriched in the regulatory sequences of Oct4, B-Globin and Thy1, correlating with the repressed state of these genes in these cells. MacroH2A.1 did not become enriched in the GAPDH and c-Jun promoter, active in all cell types analyzed (Fig. 3). MacroH2A.1 was highly enriched in the chromatin of Sox2 regulatory regions in MEFs but not in NSCs in which this gene is highly expressed. In conclusion, the histone variant macroH2A.1 incorporates into the nucleosomes in the regulatory sequences of both repressed pluripotency and lineage-specific genes in somatic cells linking macroH2A not only to gene silencing but also to the stabilization of the differentiated cell state.

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