Dynamic chromatin states in human ES cells reveal potential regulatory sequences and genes involved in pluripotency

Abstract

Pluripotency, the ability of a cell to differentiate and give rise to all embryonic lineages, defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes, accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells, as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency, we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation, except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression, suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers, and observed much greater dynamics in chromatin modifications, especially H3K4me1 and H3K27ac, which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

Figure 1

Dynamic switch of H3K27 modifications at promoters. (A; left) Heat map of histone modifications H3K4me1, H3K4me3, H3K27ac and H3K27me3 within 5 kb of 22 047 TSSs, before and after differentiation. Middle: for each gene and cell type, we calculate the difference (H3K27ac – H3K27me3), and rank genes by comparing the difference of this value between the cell types (DFC – hESC). A negative value represents hESC enrichment of H3K27ac and DFC enrichment of H3K27me3 (blue C_g). A positive value represents DFC enrichment of H3K27ac and hESC enrichment of H3K27me3 (red C_g). Right: difference in gene expression (DFC/hESC); blue is hESC-specific expression while red is DFC-specific expression. Representative genes are noted on the far right. (B) UCSC Genome Browser snapshots showing the log2 ratio enrichment for H3K27ac (red), H3K27me3 (green) and H3K4me3 (orange) compared to input. Gene names are listed at the 5′ end of the gene structure. Left: a 10-kb window around the HAND1 gene illustrating the presence of H3K27me3 in hESCs that switches to H3K27ac following differentiation. (right) A 14.3-kb window around the SOX2 gene illustrating the presence of H3K27ac in hESCs that switches to H3K27me3 following differentiation.

Figure 2

Enhancer features and cell-type specificity. (A; left) A heat map of histone modifications ± 5 kb of predicted enhancers, ranked based on differences in H3K27ac (DFC – hESC). Middle: the cell-type specificity of chromatin modifications at enhancers, C_e = (H3K27ac_DFC – H3K27ac_hESC). Right: changes in gene expression of neighboring genes. (B) UCSC Genome Browser snapshots of a 188-kb window at the FOXD3 locus showing the log2 ratio enrichment for H3K27ac (red), H3K27me3 (green), H3K4me3 (orange), H3K4me1 (blue) and CTCF (purple-dashed line indicates binding outside gene) compared to input. Predicted enhancers (blue bars above H3K4me1 peaks) lose their chromatin signature after differentiation. (C) Distribution of enhancers in each cell type relative to 5′ and 3′ ends of genes, as well as intragenic and intergenic regions. (D) Overlap of ChIP-Seq binding sites for transcription factors SOX2 and NANOG, compared to promoters, predicted hESC enhancers and predicted DFC enhancers.

Figure 3

Enrichment of cell-specific enhancers within CTCF-organized regulatory domain (CORDs) and enhancer validation. (A) Diagram of CORDs. Regions bounded by CTCF containing promoters and enhancers. (B) Distribution of hESC specific, DFC specific and nonspecific enhancers within CTCF-defined domains containing promoters of hESC-specific, DFC-specific, and nonspecific genes. (C) As in B, but expanded to neighboring CTCF-defined domains. (D) Reporter assays of enhancer function at predicted hESC enhancers and randomly chosen genomic regions, cloned downstream of a luciferase gene. The dashed red line indicates a P-value cutoff of 1%. hESC-specific enhancers were selected from within CORDs for known ES-related genes and tested in H1 and HeLa cells. H1-H3, enhancers specifically marked in HeLa cells; N/A, failed transfection in HeLa. (E) 3C was performed to assess the interactions between the FOXD3 promoter and three predicted enhancers in its CTCF-defined domain (E1, E2 and E3). The interaction strength is compared to predicted enhancers outside the CTCF-defined domain (CE1 and CE7), to loci lacking the enhancer chromatin signature (C2-C6), control regions on a different chromosome (S_C1-S_C3) and water.

Figure 4

Subset of shared enhancers are poised for early response. (A) Enhancer enrichment relative to gene expression for three subsets of enhancers: those uniquely marked in hESCs (blue), those uniquely marked in DFCs (orange) and the remaining 8 863 that are marked in both (grey). A subset of shared enhancers is enriched at differentially expressed genes in both cell types (see Supplementary information, Data S1). (B) UCSC Genome Browser snapshots of MSX1 and MEIS1 gene loci. These genes are specifically expressed in DFCs, but have H3K4me1-marked enhancers in hESCs (blue). These enhancers lack H3K27ac (red) in hESCs, which is highly enriched following differentiation. (C, D) We measured gene expression at 3, 6, 12, 24, 48, 72 and 120 h after BMP4/bFGF treatment of hESCs. For differentially expressed genes at each time point, we counted the average number of acetylated enhancers with cell-type specificity, defined as the 2 000 shared enhancers with the most H3K27ac in (C) DFCs and (D) hESCs. Random is described in the Supplementary information, Data S1.

Figure 5

Model of cell-type-specific enhancers and poised enhancers in cell fate. This model illustrates the role of poised enhancers in hESC pluripotency and cell fate commitment. ES cells grown in the presence of BMP4 and bFGF give rise to three of four possible lineages (ectoderm excluded). Poised enhancers contribute to initiation of lineage determination by activating early response genes that go on to establish the cell fate.