A unique chromatin signature uncovers early developmental enhancers in humans

Abstract

Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term 'poised enhancers', are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.

Figure 1. Unique chromatin signatures distinguish two classes of regulatory elements in hESCs

a, Genome browser representations of p300, H3K4me1, H3K27ac, H3K27me3 and H3K4me3 enrichment profiles in hESCs are shown for a representative class I (for example, NANOG, top) and class II (for example, NODAL, bottom) element and its flanking regions. The peak height corresponds to normalized fold enrichments as calculated by QuEST. b, Average hESC ChIP-seq signal profiles were generated for the indicated histone modifications around the central position of p300-bound regions, over class I (top) and class II (bottom) elements, respectively. c, Class I and II elements were mapped to their closest Ensembl gene TSS and the distribution of distances between elements and TSS is shown.

Figure 2. Functional and molecular characterization of class I and II elements

a, b, Sequential ChIP experiments were performed from hESCs with the indicated pairs of histone modification antibodies. ChIP material was analysed by qPCR for select class I and class II elements, as well as negative control regions (NEG1–3). The y axis shows per cent input recovery; error bars represent standard deviation (s.d.) from three technical replicates. c, RNA-seq data set was obtained from hESC poly(A)-RNA and reads per kilobase per million mapped reads (RPKM) were calculated for all human Ensembl genes. RPKMs for all annotated genes (green) or for those closest to class I (red) or class II (blue) elements are represented as box plots. P-values were calculated using non-paired Wilcoxon tests. In the box plots, bottom and top of the boxes correspond to the 25th and 75th percentiles and the internal band is the 50th percentile (median). The plot whiskers extending outside the boxes correspond to the lowest and highest datum within 1.5 interquartile range of the lower and upper quartiles, respectively. d, e, Functional annotation of class I (d) and class II (e) elements was performed using GREAT. The top over-represented categories belonging to three different ontologies are shown: Mouse Genome Informatics (MGI) expression detected (red) contains information on tissue- and developmental-stage-specific expression in mouse; Gene Ontology (GO) biological process (green) describes the biological processes associated with gene function; mouse phenotypes (blue) ontology contains data about mouse genotype–phenotype associations. The x axes values (in logarithmic scale) correspond to the binomial raw (uncorrected) P-values.

Figure 3. A subset of class II elements acquires active enhancer chromatin signature upon neuroectodermal differentiation

a, Average hNEC ChIP-seq signal profiles were generated for the indicated histone modifications around the central position of those p300-bound regions (as determined in hESC) that acquired H3K27ac enrichment in hNECs (that is, class II→I elements). b, Genome browser representation of p300, H3K4me1, H3K27ac and H3K27me3 (in hESCs and hNECs) binding profiles at a representative class II→I element. The peak height corresponds to normalized fold enrichments as calculated by QuEST. c–e, ChIP-qPCR analyses from hNECs with indicated histone modification antibodies at select elements including: class I elements that were only active in hESCs (active ESC), or in both hESCs and hNECs (active both), or class II elements that did not acquire H3K27ac in hNEC (class II), or class II→I elements. The y axis shows per cent input recovery; error bars represent s.d. from three technical replicates. ChIPs used in these qPCRs represent biological replicates of those samples used in ChIP-seq. f, RNA-seq data sets from hESC and hNEC poly(A)-RNA were used to calculate the RPKM for all human Ensembl genes. RPKMs in both cell types are represented as box plots for all genes (All), genes linked to class I elements, genes linked to class II elements, and genes linked to class II→I elements. P-values were calculated using paired (NEC class II→I versus ESC class II→I) or non-paired (NEC class II→I versus NEC class II) Wilcoxon tests.

Figure 4. Class II elements have developmental enhancer activity in vivo

a, Merged bright-field and GFP images are shown for representative shield stage zebrafish embryos injected with class II elements proximal to human EOMES, LEFTY2 and NODAL. For the EOMES enhancer, dorsal (anterior to top) and lateral (shield to right) views are presented in the left and right panels, respectively. For LEFTY2 and NODAL, animal pole (shield to top) and lateral (shield to right) views are presented in the left and right panels, respectively. White arrows indicate the location of the shield in each image. A, anterior; D, dorsal. Scale bar, 150 μm. b–f, Merged bright-field and GFP images are shown for representative 24–28 h.p.f. zebrafish embryos injected with class II elements proximal to SOX2 (b), EN1 (c), NKX2-1 (d), WNT8B (e) and MIXL1 (f) genes. In b–e, schematics highlighting the relevant anatomical structures where GFP expression was reproducibly observed are shown on the left, and three images correspond, from left to right and top to bottom, to whole-embryo flattened dorsal views, dorsal anterior views and lateral anterior views, respectively. In f, a lateral posterior view is shown. In b–f, scale bar = 150 μm. MHB, midbrain–hindbrain boundary. g, Proposed model for enhancer bookmarking during early embryonic development. Poised developmental enhancers (class II) are marked by a unique chromatin signature, involving occupancy of chromatin modifiers p300, BRG1 and PRC2 and nucleosomal regions marked by H3K4me1 and H3K27me3. During differentiation, appropriate developmental and signalling cues are able to rapidly transition these poised, pre-marked enhancers into an active state represented by the acquisition of H3K27ac, RNA POL2 binding, recruitment of tissue-specific transcription factors (TFs) and loss of H3K27me3, leading to the establishment of tissue-specific gene expression patterns.