Erythropoietin (EPO) regulates expression of genes that drive proliferation, survival, and differentiation of erythroid progenitor cells into mature erythrocytes. The EPO receptor signals primarily via the JAK2-STAT5 pathway. Only a few direct target genes of this pathway have been identified to date.
To find novel EPO-induced target genes in human erythroid cells (HUDEP2 cells), we employed a multi-omics approach. Three hours of EPO starvation followed by stimulation for 1 hour resulted in rapid phosphorylation of STAT5, but not STAT1 or STAT3. ChIP-seq for pSTAT5 identified 3128 binding sites. The majority of peaks contain a palindromic ‘GAS’ motif (TTCYXRGAA), and are located at intronic (50%), distal (29%) and intergenic (15%) enhancers; only 3% are located at promoters. De novo motif discovery identified significant enrichment of DNA-binding motifs for GATA and KLF transcription factors (TFs), suggesting co-operativity between EPO signalling and the essential basal erythroid TFs, GATA1 and KLF1.
To examine STAT5-independent changes in chromatin, we performed differential analysis of ATAC-seq peaks before and after EPO stimulation. We found 14,535 EPO-responsive regions, whereas only ~8% overlap with pSTAT5 ChIP-seq peaks, suggesting EPO-mediated phosphorylation and DNA-binding of undiscovered TFs. De novo motif discovery analysis within these regions discovered binding sites for TFs of the NFY, EGR, and NRF families, suggesting a hidden complexity of TFs that mediate responses to EPO.
We developed Body-SLAM-seq, a novel metabolic labelling technique of primary RNA-seq based on SLAM-seq, to determine the immediate transcriptional targets of EPO. We found ~100 differentially transcribed genes (DTGs) in immediate response to EPO. Some are direct pSTAT5 target genes (e.g. BCL2L1, PIM1, and CISH); others are immediate early genes that are involved in transcriptional regulation and feedback signalling (e.g. EGR1, OSM and DUSP6).