Identification of cis regulatory features in the embryonic zebrafish genome through large-scale profiling of H3K4me1 and H3K4me3 binding sites

Abstract

An organism's genome sequence serves as a blueprint for the proteins and regulatory RNAs essential for cellular function. The genome also harbors cis-acting non-coding sequences that control gene expression and are essential to coordinate regulatory programs during embryonic development. However, the genome sequence is largely identical between cell types within a multi-cellular organism indicating that factors such as DNA accessibility and chromatin structure play a crucial role in governing cell-specific gene expression. Recent studies have identified particular chromatin modifications that define functionally distinct cis regulatory elements. Among these are forms of histone 3 that are mono- or tri-methylated at lysine 4 (H3K4me1 or H3K4me3, respectively), which bind preferentially to promoter and enhancer elements in the mammalian genome. In this work, we investigated whether these modified histones could similarly identify cis regulatory elements within the zebrafish genome. By applying chromatin immunoprecipitation followed by deep sequencing, we find that H3K4me1 and H3K4me3 are enriched at transcriptional start sites in the genome of the developing zebrafish embryo and that this association correlates with gene expression. We further find that these modifications associate with distal non-coding conserved elements, including known active enhancers. Finally, we demonstrate that it is possible to utilize H3K4me1 and H3K4me3 binding profiles in combination with available expression data to computationally identify relevant cis regulatory sequences flanking syn-expressed genes in the developing embryo. Taken together, our results indicate that H3K4me1 and H3K4me3 generally mark cis regulatory elements within the zebrafish genome and indicate that further characterization of the zebrafish using this approach will prove valuable in defining transcriptional networks in this model system.

Fig. 1

H3K4me1 and H3K4me3 binding sites in the zebrafish genome. A–C, Screenshots from a mirrored UCSC Genome Brower displaying enriched H3K4me1 and H3K4me3 regions (hotspots), peaks within these regions, sequence tag density (signal), annotated ENSEMBL and/or RefSeq genes, and expressed sequence tags (B and C only). A. Region of zebrafish chromosome 14 containing the lnx2b, chic1, cdx4, and kdrl genes. B. 5′ end of the lnx2b gene. C. 5′ end of the tal1 gene.

Fig. 2

Genome-wide distribution of H3K4me1 and H3K4me3 binding sites in relation to transcriptional start sites (TSS). A, D. Density plots of H3K4me1 and H3K4me3 binding at a distance of 100,000 nucleotides up and downstream of ENSEMBL (A) or RefSeq (D) TSSs. B, C, E, F. Histograms showing frequency of peaks at an interval of 10,000 nucleotides up and downstream of TSSs for ENSEMBL (B, C) or RefSeq (E, F). B and E, Distribution of H3K4me3 peaks. C, F. Distribution of H3K4me1 peaks.

Fig. 3

Correlation of H3K4me1 and H3K4me3 TSS occupancy with expression level. A, B. Graphs representing proportion of ENSEMBL (A) or RefSeq (B) genes with indicated expression level that are unbound or bound by H3K4me1, H3K4me3, or both at their TSS. “Vasc” refers to genes expressed in blood vessels based on available whole mount in situ hybridization data.

Fig. 4

Association of H3K4me1 and H3K4me3 with non-coding conserved elements (NCEs) and known enhancers in the zebrafish genome. A, B. Distribution of H3K4me1 (A) or H3K4me3 (B) in relation to distal NCEs. C. The zebrafish hoxb4a locus. D. The zebrafish fgf8a locus. C, D. Enriched H3K4me1 and H3K4me3 binding regions (hotspots), peaks within these regions, sequence tag density (signal), and annotated ENSEMBL and/or RefSeq genes are shown. The activity of functionally validated CNEs is indicated by the color code legend in (C).

Fig. 5

H3K4me1-positive elements can be active enhancers in zebrafish embryos. A. Left, genomic region upstream of notch3 showing location of H3K4me1-positive element (in red) used for reporter assay. Right, expression of Egfp driven by enhancer element shown at left in hypochord (arrow), floor plate (arrowhead), and lower levels in the notochord (nc, low-level positive cells indicated by bracket); lateral view, dorsal is up, anterior to the left. B. Left, genomic region upstream of her6 showing location of H3K4me1-positive elements used for reporter assay; red and gray boxes indicate element that drove or did not drive expression, respectively. Right, expression of Egfp driven by the active enhancer element shown at left in region of the pharyngeal arches (boxed area); inset, higher magnification of pharyngeal arch region showing positive mesenchymal surrounding the arch (bracket); images are not from the same embryo lateral view, dorsal is up, anterior to the left. C. Left, genomic region upstream of dll4 showing location of H3K4me1-positive element used for reporter assay. Red and gray boxes indicate element that drove or did not drive expression, respectively. Right, expression of Egfp driven by active enhancer element shown at left in cells residing in the olfactory placode (arrows); frontal view, dorsal is up.