Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo

Abstract

Developmental regulatory genes have both activating (H3K4me3) and repressive (H3K27me3) histone modifications in embryonic stem cells (ESCs). This bivalent configuration is thought to maintain lineage commitment programs in a poised state. However, establishing physiological relevance has been complicated by the high number of cells required for chromatin immunoprecipitation (ChIP). We developed a low-cell-number chromatin immunoprecipitation (low-cell ChIP) protocol to investigate the chromatin of mouse primordial germ cells (PGCs). Genome-wide analysis of embryonic day 11.5 (E11.5) PGCs revealed H3K4me3/H3K27me3 bivalent domains highly enriched at developmental regulatory genes in a manner remarkably similar to ESCs. Developmental regulators remain bivalent and transcriptionally silent through the initiation of sexual differentiation at E13.5. We also identified >2,500 "orphan" bivalent domains that are distal to known genes and expressed in a tissue-specific manner but silent in PGCs. Our results demonstrate the existence of bivalent domains in the germline and raise the possibility that the somatic program is continuously maintained as bivalent, potentially imparting transgenerational epigenetic inheritance.

Figure 1. Bivalent genes in E11.5 PGCs are enriched for developmental regulators

(A) Sample UCSC Genome Browser view of ChIP-Seq signal for 2 biological replicates of E11.5 PGCs and for ESCs. (B) E11.5 PGCs and ESCs have a similar number of genes marked by only H3K4me3, only H3K27me3, or both. (C) Overlap of genes marked by H3K4me3 and H3K27me3 in E11.5 PGCs and ESCs, p<2.2×10⁻¹⁶ using Fisher’s exact test. (D) Top 5 biological process gene ontology terms as determined using DAVID for genes marked as bivalent in both PGCs and ESCs. (E) Validation of ChIP-Seq using ChIP-qPCR enrichment for H3K4me3 and H3K27me3 in E11.5 PGCs for a subset of bivalent genes representing regulators of the three germ layers. Data are mean percent of input ± standard deviation of enrichment. The dotted line represents level of background enrichment. (F) Heat map showing expression of the subset of genes marked as bivalent in both PGCs and ESCs in indicated cell types. EBs: embryonic bodies; MEFs: mouse embryonic fibroblasts; L.V. Brain: lateral ventricles of adult brain. Blue indicates down-regulation, red indicates up-regulation. See also Figure S2, Table S1.

Figure 2. PGCs are enriched for bivalent developmental regulators compared to surrounding somatic cells at E11.5

(A) ChIP enrichment for H3K4me3 and H3K27me3 in ESCs at the indicated genes. Data are mean percent of input ± standard deviation of enrichment. The dotted line represents level of background enrichment. (B) ChIP enrichment for H3K4me3 and H3K27me3 in E11.5 soma at the indicated genes as described in (A). (C) The ratio of H3K4me3 to H3K27me3 log₂ transformed for the indicated gene for ESCs, E11.5 PGCs, and E11.5 soma. The gray area indicates a ratio less than 2 and greater than 0.5. Genes where enrichment for either H3K4me3 or H3K27me3 is not above background are lighter in color. (D) The log₂ of the H3K4me3/H3K27me3 ratio was calculated as in (C) (log₂ R_{cell type}) for all genes examined independent of enrichment over background. The differences between cell type ratios were calculated as follows: Δ_PGC=|log₂R_PGC − log₂R_ESC|, and Δ_soma=|log₂R_soma − log₂R_ESC| for each gene. Box-plots represent the distribution of the differences. Statistical significance was assessed using the Wilcoxon matched-pairs signed rank test (1-sided), **p=0.001. ND= not determined.

Figure 3. Developmental regulators remain bivalent and transcriptionally repressed during sexual differentiation in the germline

Developmental genes remain bivalent and transcriptionally silent from E11.5 to E13.5 in both the male and female germline. ChIP enrichment for H3K4me3 and H3K27me3 in E11.5-13.5 PGCs at the indicated genes is represented as the mean percent of input ± standard deviation for at least 2 biological replicates. Transcription was measured with qRT-PCR for the indicated genes and represented as a percent of the housekeeping gene L7 ± standard deviation. At all stages of PGC development analyzed, somatic developmental genes have a similar level of enrichment for H3K4me3 and H3K27me3 and very low expression. See also Figure S3.

Figure 4. Orphan bivalent domains overlap CpG Islands and show tissue specific expression

(A) Distances of genic (assigned to gene, dark grey bars) and orphan PGC bivalent domains (white bars) to nearest gene, with transparent overlay. Distances are presented as the log base 10 of the genomic length in base pairs from bivalent domains to either their nearest gene, with bivalent domains upstream of their nearest gene assigned negative distances after logarithm. Bivalent domains occurring within a gene were assigned a genomic length of 1. TSSs were defined as the starting positions of RefSeq genes (B) Number of orphan bivalent domains in E11.5 PGCs with characteristics of transcription start site activity: occurring in unmethylated CpG islands (CGI) identified using CAP-Seq (Illingworth et al., 2010), with a CAGE expression tag in 1 or more of 22 tissue types (Kawaji et al., 2009), or both. (C) Distributions of the tissue-specificity scores of CAGE tag cluster expression in orphan bivalent domains with CGI in PGC, orphan bivalent domains without CGI in PGC, bivalent RefSeq genes, and all RefSeq genes. The p-values are from the Wilcoxon rank sum test. (D) UCSC Genome Browser view of an orphan bivalent domain in PGCs and ESCs (Mikkelsen et al., 2007) that overlaps a CGI (Illingworth et al., 2010) and a putative non-coding RNA (Guttman et al., 2009). RNA-Seq data indicates this region is transcriptionally silent in PGCs (Seisenberger et al., 2012) and ESCs (Marks et al., 2012), while CAGE tag data indicates expression in the hippocampus. (E) Hierarchical clustering of tissues based on the binary (on or off) expression status of CAGE tag clusters in regions marked as orphan bivalent domains in PGC. Yellow means expression and blue mean no expression. See also Table S2, S3.