Epigenomic analysis reveals DNA motifs regulating histone modifications in human and mouse

Abstract

Histones are modified by enzymes that act in a locus, cell-type, and developmental stage-specific manner. The recruitment of enzymes to chromatin is regulated at multiple levels, including interaction with sequence-specific DNA-binding factors. However, the DNA-binding specificity of the regulatory factors that orchestrate specific histone modifications has not been broadly mapped. We have analyzed 6 histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3, K3H9me3, H3K36me3) across 121 human cell types and tissues from the NIH Roadmap Epigenomics Project as well as 8 histone marks (with addition of H3K4me2 and H3K9ac) from the mouse ENCODE Consortium. We have identified 361 and 369 DNA motifs in human and mouse, respectively, that are the most predictive of each histone mark. Interestingly, 107 human motifs are conserved between the two species. In human embryonic cell line H1, we mutated only the found DNA motifs at particular loci and the significant reduction of H3K27ac levels validated the regulatory roles of the perturbed motifs. The functionality of these motifs was also supported by the evidence that histone-associated motifs, especially H3K4me3 motifs, significantly overlap with the expression of quantitative trait loci SNPs in cancer patients more than the known and random motifs. Furthermore, we observed possible feedbacks to control chromatin dynamics as the found motifs appear in the promoters or enhancers associated with various histone modification enzymes. These results pave the way toward revealing the molecular mechanisms of epigenetic events, such as histone modification dynamics and epigenetic priming.

Keywords: CRISPR; chromatin dynamics; cis-regulatory elements; epigenomics; locus specificity.

Fig. 1.

Identification of DNA motifs associated with six histone modifications in 121 human cells and tissues. (A, Left) The workflow of identifying motifs associated with histone modifications. (Right) The average AUCs for each mark across all 121 human cell types. (B) Association of the final motif set with the histone marks across all 121 human cell types. The x axis represents each motif cluster in the final set, color-coded by their associated histone marks. The y axis represents the ChIP-seq experiments ordered by histone modifications. Black spots inside the matrix show whether a motif cluster was found in a ChIP-seq experiment. (C) Number of motif clusters per type. (D) Example of de novo motifs matched to the known motifs.

Fig. 2.

Experimental validation of the found motifs on regulating histone modifications. (A) CRISPR and donor constructs: MfeI restriction enzyme site indicated for gRNA cloning. (B) Schematic flowchart of CRISPR-mediated knockin in H1 ESCs. (C) ChIP-qPCR results of chr1 locus: H3K27ac ChIP-seq peaks of the region are shown (Upper); WT, loxP-control, and mutant alleles are shown (Lower) with WT motifs marked in red and sequence-shuffled motifs marked in yellow; black lines indicate the survey region of ChIP-qPCR; ChIP-qPCR results are shown at the bottom, error bar is shown for three biological replicates with *P = 0.0371 for probe #1 and **P = 0.0072 for probe #2. (D) ChIP-qPCR result of chr3 locus: H3K27ac ChIP-seq peaks of the region are shown (Upper); WT, loxP-control, and mutant alleles are shown (Lower) with WT motifs marked in red and sequence-shuffled motifs marked in yellow; black lines indicate the survey region of ChIP-qPCR; ChIP-qPCR results are shown at the bottom, error bar is shown for three biological replicates with **P = 0.0026 for probe #1 and **P = 0.002872 for probe #2.

Fig. 3.

Comparison of histone-associated motifs in human and mouse. (A) Distribution of conserved/nonconserved histone motifs between human and mouse. Among conserved motifs, there are three categories: motifs that retain histone mark labels between human and mouse (Same), motifs that have similar histone mark labels between human and mouse (Similar), and motifs that have different histone mark labels between human and mouse (Different). (B) Average PhastCons scores around promoters and some example known motifs’ loci. (C, Left) Average Phastcons scores around histone motifs’ loci. (Right) Example genome browser view of human histone motif loci and PhastCons scores.

Fig. 4.

Histone-associated motifs tend to overlap with eQTL SNPs in cancer patients. (A) Distribution of TCGA SNPs and histone motifs over gene body. Each gene’s body is split into 10 equal bins. (B) Distribution of log(OPKM) of different histone motif types compared with known motifs and random motifs. (C) Log(OPKM) of histone motif and known motif per cancer type. (D) Example of motifs with highest average log(OPKM).

Fig. 5.

Histone modification can be regulated by positive or negative feedback loops. (A) Positive feedback loops in human H3K4me3. (B) Negative feedback loops in human H3K27ac. (C) Positive feedback loops in mouse H3K4me3. (D) Negative feedback loops in mouse H3K9ac. Histone signals for genome browser views are from H1 cell for human and Forebrain-E11.5 sample for mouse. Green arrows denote positive regulation, red arrows are for negative regulation, gray lines indicate association between motifs and genes (motif occurring in the promoters or enhancers of a gene). (E) Average number of H3K4me3 motif matches within promoter regions of histone methylation genes and other protein coding genes. Promoter regions are defined as 1,000-bp centered at TSS. Only protein-coding genes were considered. Genes that do not have any matches were not included. The two distributions of number of matches are greatly different from each other; Mann–Whitney U test gives a P value of 1.472 × 10⁻⁶. *n is number of H3K4me3 motif matches per gene’s promoter.