STAT5 Facilitates Terminal Erythroid Maturation in Concert with the Integrator Complex

Erythropoiesis is a remarkable biological process that occurs continuously throughout an individual's lifetime, generating over 2 million erythrocytes per second in human adults. This process is orchestrated by a complex interplay of transcription factors, epigenetic regulators, and signaling pathways that fine-tune gene expression at each stage of maturation. Our previous work has demonstrated that during terminal erythroid maturation RNA Polymerase II (RNAPII) levels decline dramatically, becoming a scarce resource allocated primarily to erythroid genes. We further demonstrated that at many loci, RNAPII becomes highly paused before RNAPII occupancy is lost (Murphy Blood 2021; Lv Blood 2023). The Integrator Complex (INT) is a modular multi-subunit machinery that is highly expressed in erythroid cells. INT binds to paused RNAPII and promotes promoter-proximal transcriptional termination through dephosphorylation of RNAPII via INT subunit 8 and cleavage of the nascent RNA transcript via INT subunit 11. Our previous genomic studies revealed an increase in the occupancy of integrator subunit 8 (INTS8) at the promoters of genes that become repressed during the terminal maturation of erythroid cells derived from CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, disruption of INTS8 in both HUDEP2 cells and erythroid-directed CD34+ HSPC cultures significantly impaired erythroid proliferation and viability, and delayed erythroid maturation. We further observed that INT binding sites in orthochromatic erythroblasts are highly enriched for STAT5 (Signal Transducer and Activator of Transcription 5) motifs, and immunofluorescence and confocal imaging demonstrated colocalization of STAT5 and INTS8, suggesting a functional interaction between STAT5 and the INT complex.

In this study, we investigated the role of STAT5 in facilitating terminal erythroid maturation via the INT complex. STAT5 is activated by erythropoietin (EPO) signaling and has been traditionally viewed as an essential factor for erythroid progenitor survival and proliferation, though its functions in later stages of erythroid maturation remain less well understood. To test the hypothesis that STAT5 cooperates with INT to regulate erythroid gene expression, we first performed CUT&RUN analysis for INTS8 and phosphorylated STAT5 (pSTAT5) in basophilic and orthochromatic erythroblasts derived from CD34+ HSPCs. Our studies revealed that genes that become repressed during terminal erythroid maturation gain both pSTAT5 and INTS8 at their promoters. In addition, these regions lose threnine1525 phosphorylated RNAPII , which indicates increased INT activity. Importantly, gain of STAT5 directly correlated with increased INTS8 occupancy, loss of elongating RNAPII, and decreased mRNA expression, indicating that STAT5 might facilitate gene repression through enhancing INT occupancy.

To functionally test the role of STAT5 in repressing gene transcription, we overexpressed (OE) STAT5 in HUDEP-2 (Human Umbilical Cord Derived Erythroid) cells. This resulted in accelerated terminal maturation, evidenced by increased red color, smaller cell size, and higher benzidine staining levels compared to empty vector controls. CUT&RUN studies indicated that STAT5 OE led to increased INTS8 occupancy at pSTAT5 binding sites, and was correlated with decreased mRNA expression, indicating that STAT5 facilitates INT occupancy to remove RNAPII and shut down transcription during terminal erythroid maturation. STAT5 OE also resulted in the upregulation of many genes, including HBB and heme synthesis genes. The majority of unregulated genes did not gain INTS8 in STAT5 OE cells. Unexpectedly, pathway analyses of the upregulated genes revealed multiple pathways related to autophagy. Further inspection of the differentially expressed genes identified significant upregulation of multiple regulators of mitophagy, including BNIP3L and ULK1. STAT5 immunoprecipitation followed by mass spectrometry revealed significant interactions with numerous mitochondrial proteins, consistent with its recently established role in mitochondrial clearance (Zhang 2021). Together, these findings suggest a dual role for STAT5 in terminal erythroid maturation, working with INT to repress non-erythroid genes while also promoting mitophagy, and provide novel insights into the regulation of terminal erythropoiesis.