Identification of Erythroid Specific Enhancers By Analyzing Genome Wide Association of Transcriptional Co-Activators CBP and P300

Enhancers are cruicial for gene regulation and demonstrate tissue, cell and developmental stage specificity. Recent studies have shown that analyzing genome wide association of CBP and p300 predicts the tissue spcific transcriptional enhancers in various tissues. Our aim was to study the transcriptional enhancers in haematopoietic stem cells and erythroid cells to identify the role of these genomic regions in determining transcriptional regulation of haematopoietic stem cell maintenance and erythroid differentiation. We obtained CD34+ cells from normal healthy donors after magnetic activated cell sorting from peripheral blood mononuclear cells. The purified CD34+ cells were differentiated by ex vivo erythropoiesis using a serum-free two-phase liquid culture system. Cells were collected at 2 different time points, before (day 5 in culture) and after differentiation (day13 in culture). ChIP assays were carried out using antibodies against CBP and p300 and two independent libraries were created using Illumina TrueSeq library kit as per the manufacturer's instruction. The libraries were sequenced in Illumina HiSeq producing 50 bp single end reads. A control sample (without antibody) was run as input as a background control. Sequenced reads were mapped to the human genome (UCSC Genome Browser hg19). To identify ChIP-Seq peaks, Model-based Alignment of ChIP-Seq (MACS) program was used with a p value of <10e-5 and enrichment factor > 5. Localization of the binding sites relative to the annotated genes and co-localization of CBP/p300 were determined using the ChIP seek analyzer. Validation of co-occupancy at selected regions was performed by quantitative-PCR analysis.

Genome wide analysis of CBP and p300 enrichment showed these co-factors are highly enriched in introns and intergenic regions than the gene promoters suggesting that these co-activators efficiently mark enhancers in the haematopoietic stem cells and erythroid cells. Co-localization of CBP/p300 with erythroid transcription factors (GATA1, KLF1, NFE2 and TAL1) was performed. The results showed that CBP/p300 are highly associated with erythroid transcription factors during differentiation indicating that transcriptional activator complexes consisting of these transcription factors and CBP/p300 in enhancer mediated transcriptional regulation in erythropoiesis. The sites of CBP/p300 occupancy were correlated with the transcriptome data and it was found that most of the top regulated genes were enriched with CBP and p300 within the intronic region in erythroid cells. We then explored whether CBP and p300 are enriched in the regions associated with DNA polymorphisms relevant to erythroid cell traits and observed that CBP/p300occupy at HBD (5'UTR), BCL11A (intron 2)and HBS1L-MYB intergenic region which contains polymorphisms linked with levels of fetal haemoglobin (HbF). We also found enrichment of these co-activators in previously mapped erythroid specific enhancers such as IKZF1, ANK1 and ABO gene loci.

Gene ontology (GO) was performed using GREAT for regions associated with CBP and p300 (binomial fold enrichment >4) and the results indicated that the genes associated with CBP were involved in erythrocyte development whereas the genes associated with p300 were found to regulate erythroid differentiation. These erythroid specific genes delineated in our study also showed conservation in mouse and were found to be associated with erythroid cell phenotypes. We also found significant enrichment of CBP and p300 in erythroid cells compared to haematopoietic stem cells for several genes that have not been previously characterized for erythroid differentiation. Taken together, our findings elucidated the roles of co-activators CBP and p300 in erythroid differentiation and further we identified these factors are enriched in previously known and new erythroid specific enhancers in association with cell specific transcription factors. Functional evaluation of the newly idenfied regulatory elements bound by CBP and p300 in the erythroid cells will provide insights in to erythroid cell development and differentiation.