Direct targets of pSTAT5 signalling in erythropoiesis

PLoS One. 2017 Jul 21;12(7):e0180922. doi: 10.1371/journal.pone.0180922. eCollection 2017.

Abstract

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.

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Line
  • Chromatin Immunoprecipitation
  • Erythropoiesis / genetics
  • Erythropoiesis / physiology*
  • Erythropoietin / genetics
  • Erythropoietin / metabolism*
  • Feedback, Physiological
  • Mice
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • STAT5 Transcription Factor / genetics
  • STAT5 Transcription Factor / metabolism*
  • Signal Transduction
  • Transcriptome

Substances

  • RNA, Messenger
  • STAT5 Transcription Factor
  • Erythropoietin

Grants and funding

The work was supported by core grant funding to A.C.P. from Mater Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.