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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.


Embryonic stem (ES), trophectoderm (TS) and extra-embryonic endoderm (XEN) cell populations have distinct replication timing profiles, which reflect their lineage potential. (A) Summary of the replication timing comparison of the selected candidate genes between the three embryo-derived stem cell lines. The replication timing of each gene was defined according to its peak abundance in G1/S1 (early, dark green), S2 (middle-early, light green), S2 and S3 (middle, yellow), S3 (middle-late, orange) or S4/G2 (late, red), determined in at least two independent experiments. Inner cell mass/ES-, TE/TS-, PrE/XEN-related loci or genes involved in the specification of somatic cell types are grouped into four different boxes. (B) Histograms comparing the relative abundance of locus-specific signal for Rex1, Sox2, Pem, Pl1, Gata6, Foxa2, Sox1 and Neurod loci within each cell cycle fraction for ES (black bars), TS (white bars) and XEN (grey bars) cells as assessed by quantitative polymerase chain reaction. Mean and standard deviation of two or more experiments are shown for each cell type analysed.
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Figure 2: Embryonic stem (ES), trophectoderm (TS) and extra-embryonic endoderm (XEN) cell populations have distinct replication timing profiles, which reflect their lineage potential. (A) Summary of the replication timing comparison of the selected candidate genes between the three embryo-derived stem cell lines. The replication timing of each gene was defined according to its peak abundance in G1/S1 (early, dark green), S2 (middle-early, light green), S2 and S3 (middle, yellow), S3 (middle-late, orange) or S4/G2 (late, red), determined in at least two independent experiments. Inner cell mass/ES-, TE/TS-, PrE/XEN-related loci or genes involved in the specification of somatic cell types are grouped into four different boxes. (B) Histograms comparing the relative abundance of locus-specific signal for Rex1, Sox2, Pem, Pl1, Gata6, Foxa2, Sox1 and Neurod loci within each cell cycle fraction for ES (black bars), TS (white bars) and XEN (grey bars) cells as assessed by quantitative polymerase chain reaction. Mean and standard deviation of two or more experiments are shown for each cell type analysed.

Mentions: In order to directly compare the epigenetic profiles of extra-embryonic stem cell lines with those of pluripotent cell lines, we initially assessed the replication timing of a panel of developmental genes in OS25, B1 and IM8A1 cell lines. Genes include those that encode transcription factors regulating the specification of germ layers in the embryo [19], as well as those encoding transcription factors that are important for the biology of early embryonic ICM, TE, PrE and EPI lineages. Replication was assessed using a previously established assay [26,27] in which asynchronous cells are pulse-labelled with 5-bromo-2-deoxyuridine (BrdU), fractionated according to cell-cycle stage (see Additional file 2, part A) and the relative abundance of newly synthesized locus-specific DNA is compared between successive cell cycle fractions using quantitative PCR. Although the exact relationship between chromatin structure and replication timing is not fully understood, early replication is a characteristic of 'accessible' and highly acetylated chromatin while late replication is a feature of heterochromatic domains and some repressed genes [28]. Consistent with this, α-globin a constitutively early replicating gene, was detected in S1 fractions isolated from ES, TS and XEN cells (Additional file 2, part B top panel), while Amylase 2.1, a late replicating control, was detected in S3 and peaked in the S4 fractions in all three cell types (Additional file 2, part B middle panel) [19]. Detection of similar levels of BrdU-labelled Gbe DNA in cell cycle fractions that were 'spiked' with a constant amount of Drosophila BrdU-labelled DNA (Additional file 2, part B lower panel), confirmed an equivalent recovery of immuno-precipitated DNA in all analyses shown. The replication times of candidate genes were determined from at least two independent experiments, scored according to a peak abundance of locus-specific DNA (in G1/S1 [early], S2 [middle-early], S2 and S3[middle], S3 [middle-late] or S4/G2 [late]) and the results were colour-coded to facilitate comparison (see Figure 2, as previously described [19,27]).


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)

Embryonic stem (ES), trophectoderm (TS) and extra-embryonic endoderm (XEN) cell populations have distinct replication timing profiles, which reflect their lineage potential. (A) Summary of the replication timing comparison of the selected candidate genes between the three embryo-derived stem cell lines. The replication timing of each gene was defined according to its peak abundance in G1/S1 (early, dark green), S2 (middle-early, light green), S2 and S3 (middle, yellow), S3 (middle-late, orange) or S4/G2 (late, red), determined in at least two independent experiments. Inner cell mass/ES-, TE/TS-, PrE/XEN-related loci or genes involved in the specification of somatic cell types are grouped into four different boxes. (B) Histograms comparing the relative abundance of locus-specific signal for Rex1, Sox2, Pem, Pl1, Gata6, Foxa2, Sox1 and Neurod loci within each cell cycle fraction for ES (black bars), TS (white bars) and XEN (grey bars) cells as assessed by quantitative polymerase chain reaction. Mean and standard deviation of two or more experiments are shown for each cell type analysed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Embryonic stem (ES), trophectoderm (TS) and extra-embryonic endoderm (XEN) cell populations have distinct replication timing profiles, which reflect their lineage potential. (A) Summary of the replication timing comparison of the selected candidate genes between the three embryo-derived stem cell lines. The replication timing of each gene was defined according to its peak abundance in G1/S1 (early, dark green), S2 (middle-early, light green), S2 and S3 (middle, yellow), S3 (middle-late, orange) or S4/G2 (late, red), determined in at least two independent experiments. Inner cell mass/ES-, TE/TS-, PrE/XEN-related loci or genes involved in the specification of somatic cell types are grouped into four different boxes. (B) Histograms comparing the relative abundance of locus-specific signal for Rex1, Sox2, Pem, Pl1, Gata6, Foxa2, Sox1 and Neurod loci within each cell cycle fraction for ES (black bars), TS (white bars) and XEN (grey bars) cells as assessed by quantitative polymerase chain reaction. Mean and standard deviation of two or more experiments are shown for each cell type analysed.
Mentions: In order to directly compare the epigenetic profiles of extra-embryonic stem cell lines with those of pluripotent cell lines, we initially assessed the replication timing of a panel of developmental genes in OS25, B1 and IM8A1 cell lines. Genes include those that encode transcription factors regulating the specification of germ layers in the embryo [19], as well as those encoding transcription factors that are important for the biology of early embryonic ICM, TE, PrE and EPI lineages. Replication was assessed using a previously established assay [26,27] in which asynchronous cells are pulse-labelled with 5-bromo-2-deoxyuridine (BrdU), fractionated according to cell-cycle stage (see Additional file 2, part A) and the relative abundance of newly synthesized locus-specific DNA is compared between successive cell cycle fractions using quantitative PCR. Although the exact relationship between chromatin structure and replication timing is not fully understood, early replication is a characteristic of 'accessible' and highly acetylated chromatin while late replication is a feature of heterochromatic domains and some repressed genes [28]. Consistent with this, α-globin a constitutively early replicating gene, was detected in S1 fractions isolated from ES, TS and XEN cells (Additional file 2, part B top panel), while Amylase 2.1, a late replicating control, was detected in S3 and peaked in the S4 fractions in all three cell types (Additional file 2, part B middle panel) [19]. Detection of similar levels of BrdU-labelled Gbe DNA in cell cycle fractions that were 'spiked' with a constant amount of Drosophila BrdU-labelled DNA (Additional file 2, part B lower panel), confirmed an equivalent recovery of immuno-precipitated DNA in all analyses shown. The replication times of candidate genes were determined from at least two independent experiments, scored according to a peak abundance of locus-specific DNA (in G1/S1 [early], S2 [middle-early], S2 and S3[middle], S3 [middle-late] or S4/G2 [late]) and the results were colour-coded to facilitate comparison (see Figure 2, as previously described [19,27]).

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.