Enhancers and Super Enhancers Are Associated With Genes That Control Phenotypic Traits In Primary Human Erythroid Cells

Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Recent studies have shown that enhancers are frequently associated with biologically relevant and disease-associated genetic variants. We hypothesized that unique sets of enhancers and super enhancers regulate gene expression in erythroid cells, a specialized cell type evolved to carry oxygen, and associated variants influence erythroid phenotypic variability. Active enhancers are part of a chromatin landscape marked by histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27Ac). A subset of enhancers, called super enhancers, important for regulating genes critical for cell-type specific identify, have been described. Super enhancers span large regions of chromatin, have domains of transcription factors (TF), significant amounts of H3K4me1 and H3K27Ac modification, and significant amounts of Mediator (MED1) occupancy, frequently with the transcriptional activator BRD4. Using ChIP-seq, genome wide maps of enhancers were constructed for H3K4me1, H3K27Ac, MED1, and BRD4 using primary human erythroid cell chromatin. These data were combined with parallel gene expression analyses determined via RNA-seq and enhancers and super enhancers identified. Cell and tissue-type specific enhancers act over distances of tens to hundreds of kilobases, thus bona fide erythroid enhancers are expected to be enriched in the genomic vicinity of genes expressed and functional in erythroid cells. Sites of occupancy of H3K4me1 were correlated with levels of gene expression in erythroid cells. To exclude gene promoters, H3K4me1 within 1kb of annotated transcriptional start sites (TSS) were excluded from analyses. Consistent with their predicted function, there was significantly higher levels of erythroid transcription for genes with H3K4me1 occupancy within 1-50kb of the TSS of genes cf. genes with H3K4me1 occupancy >50kb of a TSS (p value<2.2e-16). There was also significantly higher expression of genes with H3K4me1 occupancy within 1-50kb of the TSS in erythroid cells cf. non-erythroid cells (T lymphocyte). The top over represented TF motifs at sites of H3K4me1 were GATA1, AP1/NFE2, and KLF1. To explore whether candidate erythroid enhancers are enriched in regions associated with biologically relevant erythroid cell traits, candidate enhancers were mapped to a data set of erythroid-associated SNPs from the NHGRI GWAS catalog. 32 enhancers mapped to sites previously associated with biologically relevant erythroid traits. SNPs changed TF binding motifs at numerous enhancers including GATA1 motifs in the BCL11A, TFRC and ATP24 loci, an NFE2 motif in the ATP2B4 locus, and a TAL1 motif in the BCL11A locus. Super enhancers were identified as described (Cell 153:307, 2013) by finding regions with the highest levels of clustered chromatin modification/occupancy. Super enhancers defined by H3K4me1 and H3K27Ac modifications yielded 231 regions, BRD4 occupancy yielded 166 regions, and MED1 occupancy yielded 52 regions. H3K4me1/H3K27Ac-marked SE regions were found near the FOXO3, GATA2, STAT5A, TAL1, and ZFPM1 gene loci. BRD4- and MED1-marked super enhancers were found near the critical erythroid volume regulatory gene PIEZO1. The top over represented TF motifs at super enhancer sites defined by H3K4me1 were TAL1/RUNX1, GATA1, KLF1, defined by BRD4 were TAL1, KLF1, and MYC, and defined by MED1 were GATA1, MYC and CTCF. Mapping of super enhancers to erythroid-associated SNPs from the GWAS catalog of the NHGRI revealed many super enhancers mapped to regions associated with biologically relevant erythroid cell traits. For example, super enhancers identified by H3K4me1 mapped to loci for BCL11A, TFRC, KIT, HBS1L, MYB, ANK1, HK1, and the alpha-globin gene cluster; super enhancers identified by BRD4 localized to the alpha-globin cluster and the PIEZO1 gene locus. Perturbation of enhancer function during erythroid development and differentiation may lead to dysregulation of gene expression with concomitant phenotypic consequences. Insights into regulation of programs of gene expression in obtained from study of erythroid enhancers will provide insights into the functional significance of sequence variation associated with quantitative traits and inherited and acquired hematologic disease.