Identification of biologically relevant enhancers in human erythroid cells

Abstract

Identification of cell type-specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses, and candidate enhancers were identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the transcriptional start site (TSS) of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or phenotype. Candidate enhancers exhibited moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a set of erythroid-associated, biologically relevant, SNPs from the genome-wide association studies (GWAS) catalogue of NHGRI, National Institutes of Health. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function and provide insights into inherited and acquired hematologic disease.

FIGURE 1.

Distribution of p300 and erythroid transcription factor occupancy in human primary erythroid cell chromatin. The human genome was portioned into six bins relative to RefSeq genes. The data below represent the percentage of the human genome represented by each bin and the distribution of the p300 and erythroid transcription factor binding sites in each bin. TES, transcriptional end site.

FIGURE 2.

Gene expression and p300 and erythroid transcription factor occupancy in human primary erythroid cells. Gene expression levels were determined in primary human erythroid cell mRNA using Illumina microarrays. Expression levels of genes with p300 and erythroid transcription factor binding sites between 1 and 50 kb from the transcription start site (white boxes) were compared with the expression levels of genes with binding sites >50 kb away (gray boxes). Combinations of the four erythroid transcription factors studied (any two of the four, any three of the four, and all four) are shown. Error bars, S.D.

FIGURE 3.

Conservation plots. The average PhastCons score in the 1000 bases surrounding the center of TF-bound regions is shown. The PhastCons score is a measure of the phylogenetic conservation within mammalian genomes. Promoter (left top), exon (left bottom), intergenic (right top), and distal (right bottom) regions were analyzed separately. Intronic regions are not shown.

FIGURE 4.

Activity of candidate erythroid cell enhancers in luciferase reporter gene assays. Individual reporter gene plasmids were prepared with the biologically relevant enhancer elements, labeled Q1–Q14, corresponding to Table 4, cloned upstream of a human γ globin gene promoter-luciferase reporter gene cassette. These plasmids were transfected into human K562 cells as described. After 2 days, the cells were harvested, and luciferase activity was analyzed. Activity from test plasmids was normalized to that directed by the human γ-globin gene promoter-luciferase reporter gene control plasmid. Relative luciferase activity was expressed as that obtained from the test plasmids versus the activity obtained from the SV40 promoter-luciferase reporter gene plasmid pGL2P plasmid taking into account the transfection efficiency. The data are the means ± S.D. (error bars) of at least six independent transfection experiments. The positive control plasmid contained the β-globin gene HS2 enhancer cloned upstream of a γ-globin gene promoter-firefly luciferase reporter gene cassette. The negative control plasmid contained a promoterless luciferase reporter gene cassette.