Date Published: July 21, 2017
Publisher: Public Library of Science
Author(s): Kevin R. Gillinder, Hugh Tuckey, Charles C. Bell, Graham W. Magor, Stephen Huang, Melissa D. Ilsley, Andrew C. Perkins, Kevin D Bunting.
Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.
EPO is produced by specialised cells in the kidney in response to reduced oxygen tension. It binds to the erythropoietin receptor (EpoR) expressed on erythroid progenitor cells to induce proliferation, differentiation, survival and enucleation. Thus, an EPO-EpoR feedback loop re-establishes physiologically appropriate numbers of circulating mature red blood cells in response to need. Exogenous use of EPO, inherited mutations in the EpoR , and acquired gain-of-function mutations in JAK2 [2,3], induce supra-physiological production of red blood cells. Conversely, loss-of-function experiments have shown both EPO and EpoR are essential for the proliferation and survival of erythroid cells [4,5].