Direct Targets of Epo Receptor-JAK2-pSTAT5 Signalling in Erythropoiesis

Erythropoietin (EPO) acts through the dimeric erythropoietin receptor (EpoR) to stimulate proliferation, survival and differentiation of colony-forming units-erythroid (CFU-e). We undertook two complimentary approaches to find pSTAT5-depenendent and independent target genes in erythroid cells. We performed RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5, 30 minutes after EPO stimulation of J2E cells. This is the first time genome wide pSTAT5 ChIP-seq has been successfully undertaken in hematopoietic cells. We found ~320 robust pSTAT5 occupied sites in the erythroid genome. About 15% of these reside in promoters while the rest reside in intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of peaks contained a central palindromic GAS element, TTCYXRGAA, and there was significant enrichment of GATA and CACCC-box elements, suggesting co-regulation of some EPO-induced genes by GATA1 and KLF1. Using 4sU RNA-seq and CAGE data, we found just 57 genes to be immediately transcribed in response to EPO within 30 minutes while other target genes had delayed responses. We suggest this has biological relevance for feed forward and feedback controls on EPO driven erythropoiesis. Some of the DEGs (e.g. Bcl2l2 and Cish) are known direct targets of pSTAT5, but many are novel and suggest new pathways by which EPO regulates erythropoiesis. These include mRNA splicing, epigenetic regulation via histone methylation, and adaptive changes in the composition of the EpoR. This could provide increased sensitivity of erythroid progenitor cells to anaemic stress; i.e. sensitization of erythroid cells to JAK2-STAT5 signalling. We found a significant overlap between direct STAT5 target genes in erythroid cells, mammary epithelial cells and lymphoid cells, which imply conserved generic effectors of JAK-STAT5 signalling in different cell types. Our results provide new insights into how EPO co-ordinates erythroid cell proliferation, survival and differentiation. Some of these DEGs could be used as biomarkers for monitoring disease activity in polycythaemia vera (PV) and responses to JAK2 inhibitors.