HEXIM1 is a critical regulator of cell cycle progression and terminal maturation during steady-state and stress erythropoiesis

Abstract

Abstract Background: Erythropoiesis, the dynamic process generating approx. 2.5 million erythrocytes per second, necessitates precise transcriptional control to navigate the complex transitions from hematopoietic stem/progenitor cells (HSPCs) through distinct progenitor (BFU-E, CFU-E) and precursor stages (proerythroblast to orthochromatic erythroblast) to mature, enucleated red blood cells. While the importance of RNA Polymerase II (RNAPII) regulation is established, the specific contributions of factors governing RNAPII pausing and elongation across these developmental stages, particularly under conditions of hematopoietic stress, remain incompletely defined. Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1), regulates RNAPII by controlling the availability and activity of pTEFb (positive transcription elongation factor beta), in the context of the 7SK snRNP (small nuclear ribonucleoprotein) complex, and can either activate or repress transcription depending on genomic context. We have previously demonstrated that HEXIM1 is a key regulator of RNAPII pausing and gene expression during early and terminal erythropoiesis, as well as erythroid output in HUDEP-2 cells and in primary CD34+ HSPCs (Lv. X., Blood. 2023). We hypothesize that HEXIM1 is essential for both steady-state and stress erythropoiesis. Results: We investigated HEXIM1 function in erythropoiesis utilizing an inducible Hexim1fl/fl; Gata1[ERT2]-Cre mouse model. At steady-state, Hexim1 deletion – achieved via intraperitoneal injection of tamoxifen – resulted in impaired terminal erythroid maturation, in-vivo. Next, we investigated Hexim1 deletion in the context of anemia, which was achieved via retro-orbital bleeding of 50% of the total blood volume. Tamoxifen treated Hexim1fl/fl; Gata1[ERT2]-Cre mice had significantly fewer CD71+, Ter119+ erythroid cells in the bone marrow than tamoxifen treated Cre-ve controls 48 hours after anemic challenge, suggesting an impaired response to anemic stress. To gain insights into the mechanisms underlying these findings, we induced Hexim1 deletion in ex-vivo fetal liver cultures. Tamoxifen treatment of Hexim1fl/fl; Gata1[ERT2]-Cre cultures resulted in a dramatic reduction in cell proliferation and overall erythroid output compared to Cre-ve littermate controls. Detailed flow cytometry analysis (using surface markers CD71 and Ter119) demonstrated a significant block in terminal maturation upon Hexim1 deletion, characterized by an accumulation of immature precursors and a failure to efficiently generate Ter119high orthochromatic erythroblasts. This maturation defect was further confirmed by significantly reduced hemoglobinization, assessed via benzidine staining, and morphological abnormalities observed in cytospin preparations of Hexim1-deleted cells. Interestingly, we also observed irregularities in the cell-cycle of Hexim1-deleted cultures; to investigate this we employed a dual pulse cell cycle assay using nucleoside analogs and demonstrate that Hexim1 deletion causes S-phase perturbation – reducing S-phase exit without affecting S-phase entry. RNA-seq of CD71high, Ter119high FACS sorted cells from ex-vivo fetal liver cultures revealed 3,000 differentially expressed genes (LFC>0, p <0.05), with 1340 genes upregulated and 1660 downregulated. The downregulated genes were significantly enriched (adj. p-value <0.005) for pathways related to erythrocyte homeostasis and differentiation, including heme biosynthetic process, erythrocyte maturation, transferrin transport, and porphyrin-containing compound biosynthesis. The downregulated genes were also significantly enriched for multiple pathways related to cell cycle progression, including regulation of cell cycle, transition of G2/M transition of mitotic cell cycle, and mitotic cell cycle phase transition. Upregulated genes in GO analyses were related to immune response pathways and apoptosis. Further genomic analyses are ongoing. Conclusions: Our collective published and preliminary data strongly position HEXIM1 as a critical, multifaceted regulator essential for early progenitor expansion and late-stage erythroid maturation. Our recent studies position HEXIM1 as an essential cell cycle regulator in erythropoiesis. Future work aims to dissect the definitive physiological roles and downstream molecular consequences of Hexim1 deletion in adult mice under both steady-state and anemic stress conditions, aiming to clarify its contribution to erythropoiesis in health and disease.