HEXIM1 Regulates Early Erythropoiesis and Participates in Multiple Complexes in Erythroid Cells

In a healthy adult, steady-state erythropoiesis generates a remarkable ~2.5 million mature RBCs every second. The process of erythropoiesis can be divided into distinct phases: early erythropoiesis- the commitment of multi-lineage progenitors into erythroid progenitor cells, and terminal erythroid maturation- the maturation of proerythroblasts through several morphologically identifiable intermediates into orthochromatic erythroblasts that ultimately enucleate. We have previously demonstrated a key role for RNA Polymerase II (RNAPII) pausing in terminally maturing erythroid cells (Murphy, 2021) and have further demonstrated that Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1), is a key regulator of RNAPII pausing and gene expression during terminal erythroid maturation (Lv X, 2023). Our ongoing studies demonstrate HEXIM1 is essential for erythropoiesis in vivo, with EpoR cre-mediated deletion of HEXIM1 leading to visible anemia and lower red blood cell counts by embryonic day 16.5. Whether HEXIM1 plays a role during early erythropoiesis and the precise mechanisms by which HEXIM1 regulates erythroid gene expression remain to be fully elucidated.

Overexpression of HEXIM1 (HEXIM1 OE) in CD34⁺ hemopoietic stem and progenitor cells (HSPC), resulted in expansion of immunophenotypic erythroid progenitor (EP) populations 3 and 4, as well as functional colony-forming cells in methylcellulose, indicating that HEXIM1 also regulates early erythropoiesis. In contrast, CRISPR/Cas9 mediated disruption of HEXIM1 in erythroid-cultured CD34⁺ HSPCs resulted in impaired erythroid differentiation. 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 RNA) complex, and can either activate or repress transcription depending on genomic context. Inhibition of pTEFb with the selective inhibitor NVP-2 revealed a block in erythroid progenitor differentiation and a failure to form erythroid colonies, suggesting that HEXIM1 promotes early erythropoiesis by means of pTEFb.

To gain insights into mechanisms by which HEXIM1 regulates erythroid gene expression and function, we identified HEXIM1 binding partners in HUDEP2 cells by mass spectrometry. As expected, all members of the canonical 7SK snRNP complex were significantly enriched in the HEXIM1 pull downs, including pTEFb (a dimer of CDK9 (Log2 Fold Change(L2FC) 3.1, pValue 0.001) and CCNT1 (L2FC 5.15, pValue 0.05)), LARP7 (L2FC 4.5, pValue 0.02), and MePCE (L2FC 8.37, pValue 0.0003). HEXIM1 also pulled down TRIM28 (L2FC 2.5, pValue 0.03), which is essential for erythropoiesis (Hosoysa 2013), and has a well-described role in targeting HEXIM1 to chromatin (Espinosa, 2022). HEXIM1 participates in the 7SK complex as a homodimer or as a heterodimer with its paralog HEXIM2 (Byers, 2005). HEXIM2 was highly enriched in the HEXIM1 pull down (L2FC 6.5, pValue 0.004). HEXIM2 lacks a key regulatory domain present in HEXIM1, and has different binding affinity for pTEFb (YIK 2005). In contrast to HEXIM1, HEXIM2 is expressed most highly during early erythropoiesis and is downregulated with erythroid maturation, leaving open the possibility that HEXIM1 function shifts as heterodimers are replaced with homodimers. Intriguingly, HEXIM2 can’t fully compensate for loss of HEXIM1, and its levels are not changed following HEXIM1 OE or knockdown in erythroid cells. Functional studies on the role of HEXIM2 in early erythropoiesis are ongoing.

In addition to 7SK-related proteins, HEXIM1 interacted with multiple paraspeckle associated proteins, including EWSR1 (L2FC 3.5 pValue 0.01), PSPC1 (L2FC 2.5 pValue 0.01), DAZAP1 (L2FC 2.3 pValue 0.001), HNRNPA1 (L2FC 1.7 pValue 0.04), CPSF6 (L2FC 1.4 pValue 0.02), and SFPQ (L2FC 1.34 pValue 0.02). Paraspeckles are subnuclear bodies that have an established role in the regulation of gene expression (Taiana, 2020). RNA FISH (fluorescence in-situ hybridization) demonstrated co-localization of HEXIM1 with the short isoform of the essential paraspeckle RNA NEAT1 in both HUDEP2 cells and CD34+ derived erythroblasts, validating HEXIM1 presence in paraspeckles. Together these data indicate that HEXIM1 plays essential roles both in early erythropoiesis and in terminal erythroid maturation by utilizing multiple mechanisms to regulate stage-specific gene transcription.