Erythropoietin (EPO) regulates expression of genes that drive proliferation, survival and differentiation of erythroid progenitor cells. The EPO receptor signals via the JAK2/STAT and PI3K/MAPK pathways. Only a few direct target genes of these pathways have been identified to date. To examine EPO-induced target genes in human erythroid cells, we employed an EPO-dependent erythroid progenitor model (HUDEP-2 cells). EPO stimulation resulted in marked phosphorylation of STAT5. Analysis of ChIP-seq of 1 hour EPO stimulation identified 3128 enriched pSTAT5 binding sites; the majority of peaks contain a ‘GAS’ motif (TTCYXRGAA), and are located at intronic (50%), distal (29%) and intergenic (15%) putative enhancers; only 3% are located at promoters. De novo motif discovery identified significant enrichment of motifs for GATA and KLF transcription factors (TFs), suggesting co-operativity between external signaling (EPO) and essential erythroid TFs, GATA1 and KLF1.
To find STAT5-independent changes in chromatin state, we performed differential analysis of ATAC-seq peaks. We found 14,535 EPO-responsive regions. Most of these occur at promoters. Only ~8% overlap with pSTAT5 ChIP-seq peaks, suggesting EPO-mediated phosphorylation and DNA-binding of additional TFs. By searching for enriched motifs within these regions, we identified putative TFs of the SP/KLF, NFY, EGR, NRF1 families and CTCF. This suggests members of these families of TFs may mediate some of the biological effects of EPO.
We developed a novel metabolic labelling technique and bio-informatic pipeline, called BodySLAM-seq, to determine the immediate transcriptional targets of EPO. We used this technique to find both differentially expressed genes (DEGs) and differentially transcribed genes (DTGs) in response to EPO. We identified known direct pSTAT5 target genes such as BCL2L1, PIM1, CISH, OSM and MYC, and many novel targets including those involved in transcription regulation, and iron metabolism. Some of these could be used as biomarkers for monitoring disease activity in response to JAK inhibitors.