Direct targets of pSTAT5 signalling in erythropoiesis

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.

STAT5 is essential for IL-7–mediated viability, growth, and proliferation of T-cell acute lymphoblastic leukemia cells

“…STAT5 activating mutations, reported in T-ALL, [72][73][74] provide resistance to JAK inhibitors, which can be overcome by the Bcl-2 family inhibitor navitoclax. 74 Moreover, STAT5 induces the expression of Bcl-2 or Bcl-xL in different scenarios, [75][76][77] including downstream from IL-7 during normal mouse B-cell development, 40,78 and enforced expression of Bcl-2 in Il7r-deficient mice restores thymopoiesis in stages where IL-7 has a major prosurvival role. 79 In recent thymic emigrants, IL-7-mediated activation of STAT5 is associated with survival, whereas activation of PI3K/Akt pathway is associated with proliferation.…”

Acute Plasmodium berghei Mouse Infection Elicits Perturbed Erythropoiesis With Features That Overlap With Anemia of Chronic Disease

“…Expression of the EPO receptor peaks at the CFU-E and proerythroblast stage and decreases with further blast maturation such that by the reticulocyte stage expression is undetectable (Broudy et al, 1991). Signal transducer and activator of transcription 5 (STAT5) is a key downstream target of EPO receptor signaling, being responsible for the transcriptional activation of many key genes involved in erythropoiesis (Gillinder et al, 2017). Therefore, levels of phosphorylated STAT5 (pSTAT5) were used as a measure of receptor activation in the Ter119 + erythroblast population from the bone marrow and spleen, either directly or after EPO stimulation ex vivo.…”

What can we learn from ineffective erythropoiesis in thalassemia?

“…In response to EPO stimulation and receptor reorientation, Janus kinase 2 (JAK2), found on the intracellular part of EPOR, is rapidly phosphorylated creating docking sites for the SH2 domains of several signal transduction proteins, including STAT5 (Table 1) 13, 14 . Activated STAT5 translocates to the nucleus where it controls the expression of a subset of genes involved in proliferation, differentiation and survival of erythroid progenitors 15–18 . Although the JAK/STAT pathway is critical in promoting erythropoiesis, another signaling cascade is also activated by EPO stimulation; the PI3K-AKT pathway.…”