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Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo.

Santos J, Pereira CF, Di-Gregorio A, Spruce T, Alder O, Rodriguez T, Azuara V, Merkenschlager M, Fisher AG - Epigenetics Chromatin (2010)

Bottom Line: We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines.A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin.Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

ABSTRACT

Background: During early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE) and the primitive endoderm (PrE). Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression.

Results: In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) stem cell lines that were derived from the inner cell mass (ICM), TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3). Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation.

Conclusions: These data provide evidence that the diversification of defined embryonic and extra-embryonic lineages is accompanied by chromatin remodelling at specific loci. Stem cell lines from the ICM, TE and PrE can each dominantly reprogramme somatic cells but reset gene expression differently, reflecting their separate lineage identities and increasingly restricted developmental potentials.

No MeSH data available.


Histone modifications at the promoters of key developmental regulator genes in embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) cells. The abundance of active [histone 3 lysine 4 dimethylation (H3K4me2, yellow bars), histone 4 acetylation (H4ac, blue bars), histone 3 lysine 9 acetylation (H3K9ac, white bars)] and repressive [histone 3 lysine 27 trimethylation (H3K27me3, purple bars), histone 4 lysine 20 trimethylation (H4K20me3, red bars)] histone marks at selected loci was assessed in ES, TS and XEN cells by chromatin immunoprecipitation and quantitative polymerase chain reaction. Values are shown as the ratio of modified histone H3 to unmodified histone H3 immunoprecipitations and normalized to an abundantly expressed gene in each cell type; Oct4 in ES cells, Cdx2 in TS cells and Gata6 in XEN cells. Detected transcripts are highlighted in green while overt gene expression is shown in bold green. Primers were designed to the promoter region (100-600 bp upstream the transcriptional start site). Error bars represent the standard deviation of three independent experiments.
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Figure 3: Histone modifications at the promoters of key developmental regulator genes in embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) cells. The abundance of active [histone 3 lysine 4 dimethylation (H3K4me2, yellow bars), histone 4 acetylation (H4ac, blue bars), histone 3 lysine 9 acetylation (H3K9ac, white bars)] and repressive [histone 3 lysine 27 trimethylation (H3K27me3, purple bars), histone 4 lysine 20 trimethylation (H4K20me3, red bars)] histone marks at selected loci was assessed in ES, TS and XEN cells by chromatin immunoprecipitation and quantitative polymerase chain reaction. Values are shown as the ratio of modified histone H3 to unmodified histone H3 immunoprecipitations and normalized to an abundantly expressed gene in each cell type; Oct4 in ES cells, Cdx2 in TS cells and Gata6 in XEN cells. Detected transcripts are highlighted in green while overt gene expression is shown in bold green. Primers were designed to the promoter region (100-600 bp upstream the transcriptional start site). Error bars represent the standard deviation of three independent experiments.

Mentions: As anticipated, the promoters of many genes that are overtly expressed in ES cells (shown in bold, Figure 3, upper panel), as well as many bivalent genes (including Eomes, Fgf5, Foxd3, Mash1, Math1, Sox1 and Tbx15) were enriched for H3K4me2, and/or H3K9ac and H4ac at their promoters [19,20]. Exceptions included the promoters of Tpbpa and Pl1, two markers of differentiated trophoblast. In ES cells histone H3K27me3, a modification catalyzed by polycomb repressor complex 2 (PRC2), was abundant at the promoters of genes that were either not expressed or expressed at low levels, including TS-associated genes (Cdx2, Eomes, Pem, Psx1), PrE-associated genes (Gata6, Foxa2) and genes that are normally expressed by subsets of differentiated tissue (such as Mash1, Math1 and Neurod) (shown in purple in Figure 3). Some silent late-replicating genes showed only low levels of H3K27 trimethylation (Tpbpa and Pl1) suggesting that these genes, in contrast to bivalent genes, are not developmentally 'poised' in ES cells and may, therefore, require extensive chromatin-remodelling for correct developmental expression. Levels of promoter H4K20me3 (shown in red in Figure 3), a mark associated with mammalian pericentric heterochromatin [35], were modest in ES cells with the exception of Sox2 (an observation that is likely to reflect the fact that OS25 cells carrying Sox2 as a transgene). In TS (B1) and XEN (IM8A1) cell lines, in contrast to ES (OS25), H4K20me3 was detected at the promoters of many genes and was particularly enriched at several silent genes in XEN cells (Sox2, Foxd3, Sox1) (Figure 3, see Additional file 3 and Figure 1A for expression data). Taken as a whole these ChIP analyses suggest that, although the promoters of many development regulator genes are co-marked with histone modifications associated with active (acetylated, H3K4me2) and repressive (H3K27me3) chromatin in both ES and TS cells, this is not the case in XEN cells. Rather, in XEN cells histone marks that characterize accessible chromatin genes tend to be restricted to genes that are productively expressed at high (Gata6, Foxa2, Pem, Psx1) or moderate levels (Eomes, Fbx15, Rex1, Tbx15). The exception to this generalization is Math1 (lower panel of Figure 3 and Additional file 3), a promoter that is enriched for H3K4me2 in ES, TS and XEN cells and, therefore, appears to retain a bivalent (or poised) configuration. Although we do not currently know the cause or significance of this single observation, collectively our data suggest that the chromatin structure of many genes is different between the stem cell lines, supporting earlier proposals that epigenetic reprogramming occurs in embryonic and extra-embryonic lineages during early mouse development [17,18].


Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo.

Santos J, Pereira CF, Di-Gregorio A, Spruce T, Alder O, Rodriguez T, Azuara V, Merkenschlager M, Fisher AG - Epigenetics Chromatin (2010)

Histone modifications at the promoters of key developmental regulator genes in embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) cells. The abundance of active [histone 3 lysine 4 dimethylation (H3K4me2, yellow bars), histone 4 acetylation (H4ac, blue bars), histone 3 lysine 9 acetylation (H3K9ac, white bars)] and repressive [histone 3 lysine 27 trimethylation (H3K27me3, purple bars), histone 4 lysine 20 trimethylation (H4K20me3, red bars)] histone marks at selected loci was assessed in ES, TS and XEN cells by chromatin immunoprecipitation and quantitative polymerase chain reaction. Values are shown as the ratio of modified histone H3 to unmodified histone H3 immunoprecipitations and normalized to an abundantly expressed gene in each cell type; Oct4 in ES cells, Cdx2 in TS cells and Gata6 in XEN cells. Detected transcripts are highlighted in green while overt gene expression is shown in bold green. Primers were designed to the promoter region (100-600 bp upstream the transcriptional start site). Error bars represent the standard deviation of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: Histone modifications at the promoters of key developmental regulator genes in embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) cells. The abundance of active [histone 3 lysine 4 dimethylation (H3K4me2, yellow bars), histone 4 acetylation (H4ac, blue bars), histone 3 lysine 9 acetylation (H3K9ac, white bars)] and repressive [histone 3 lysine 27 trimethylation (H3K27me3, purple bars), histone 4 lysine 20 trimethylation (H4K20me3, red bars)] histone marks at selected loci was assessed in ES, TS and XEN cells by chromatin immunoprecipitation and quantitative polymerase chain reaction. Values are shown as the ratio of modified histone H3 to unmodified histone H3 immunoprecipitations and normalized to an abundantly expressed gene in each cell type; Oct4 in ES cells, Cdx2 in TS cells and Gata6 in XEN cells. Detected transcripts are highlighted in green while overt gene expression is shown in bold green. Primers were designed to the promoter region (100-600 bp upstream the transcriptional start site). Error bars represent the standard deviation of three independent experiments.
Mentions: As anticipated, the promoters of many genes that are overtly expressed in ES cells (shown in bold, Figure 3, upper panel), as well as many bivalent genes (including Eomes, Fgf5, Foxd3, Mash1, Math1, Sox1 and Tbx15) were enriched for H3K4me2, and/or H3K9ac and H4ac at their promoters [19,20]. Exceptions included the promoters of Tpbpa and Pl1, two markers of differentiated trophoblast. In ES cells histone H3K27me3, a modification catalyzed by polycomb repressor complex 2 (PRC2), was abundant at the promoters of genes that were either not expressed or expressed at low levels, including TS-associated genes (Cdx2, Eomes, Pem, Psx1), PrE-associated genes (Gata6, Foxa2) and genes that are normally expressed by subsets of differentiated tissue (such as Mash1, Math1 and Neurod) (shown in purple in Figure 3). Some silent late-replicating genes showed only low levels of H3K27 trimethylation (Tpbpa and Pl1) suggesting that these genes, in contrast to bivalent genes, are not developmentally 'poised' in ES cells and may, therefore, require extensive chromatin-remodelling for correct developmental expression. Levels of promoter H4K20me3 (shown in red in Figure 3), a mark associated with mammalian pericentric heterochromatin [35], were modest in ES cells with the exception of Sox2 (an observation that is likely to reflect the fact that OS25 cells carrying Sox2 as a transgene). In TS (B1) and XEN (IM8A1) cell lines, in contrast to ES (OS25), H4K20me3 was detected at the promoters of many genes and was particularly enriched at several silent genes in XEN cells (Sox2, Foxd3, Sox1) (Figure 3, see Additional file 3 and Figure 1A for expression data). Taken as a whole these ChIP analyses suggest that, although the promoters of many development regulator genes are co-marked with histone modifications associated with active (acetylated, H3K4me2) and repressive (H3K27me3) chromatin in both ES and TS cells, this is not the case in XEN cells. Rather, in XEN cells histone marks that characterize accessible chromatin genes tend to be restricted to genes that are productively expressed at high (Gata6, Foxa2, Pem, Psx1) or moderate levels (Eomes, Fbx15, Rex1, Tbx15). The exception to this generalization is Math1 (lower panel of Figure 3 and Additional file 3), a promoter that is enriched for H3K4me2 in ES, TS and XEN cells and, therefore, appears to retain a bivalent (or poised) configuration. Although we do not currently know the cause or significance of this single observation, collectively our data suggest that the chromatin structure of many genes is different between the stem cell lines, supporting earlier proposals that epigenetic reprogramming occurs in embryonic and extra-embryonic lineages during early mouse development [17,18].

Bottom Line: We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines.A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin.Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

ABSTRACT

Background: During early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE) and the primitive endoderm (PrE). Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression.

Results: In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) stem cell lines that were derived from the inner cell mass (ICM), TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3). Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation.

Conclusions: These data provide evidence that the diversification of defined embryonic and extra-embryonic lineages is accompanied by chromatin remodelling at specific loci. Stem cell lines from the ICM, TE and PrE can each dominantly reprogramme somatic cells but reset gene expression differently, reflecting their separate lineage identities and increasingly restricted developmental potentials.

No MeSH data available.