Zachary C. Murphy, Kristin E. Murphy, Michael Getman
Nov 5, 2021
Terminal erythroid maturation is associated with dramatic changes in gene expression in the setting of a cell that is undergoing rapid division and nuclear condensation. Disruption of this process is associated with inherited anemias and myelodysplastic syndromes. Recent work from our laboratory revealed that terminal erythroid maturation is associated with a dramatic decline in the level of total and elongation competent RNA polymerase II (Pol II), and that control of pol II activity is a critical step in the regulation of gene expression during terminal erythroid maturation. We further demonstrated that HEXIM1, which is highly expressed in early erythroid cells compared to most other cell types (biogps.org; bloodspot.eu), is essential for erythropoiesis (Murphy Blood 2021). The goal of our current study is to understand the mechanisms by which HEXIM1 regulates erythroid gene expression. HEXIM1 can impact gene expression though multiple mechanisms, most notably by associating with pTEFb, which is required for release of "paused" pol II into active transcription (reviewed in Michels, Transcription, 2018). HEXIM1 can inhibit transcription through sequestration of pTEFb in the 7SK ribonuclear complex, rendering it incapable of facilitating pause release. Alternatively, it can activate transcription by delivering pTEFb to target loci (McNamara Genome Data 2016). In erythroid cells, disruption of HEXIM1 impaired the expression of many erythroid specific genes, such as GYPA and many of the heme synthesis enzymes, while overexpression (OE) of HEXIM1 promoted their expression (Murphy, Blood, 2021). We therefore hypothesized that in maturing erythroblasts, HEXIM1 targets pTEFb to erythroid specific genes, promoting the establishment of appropriate patterns of gene expression and facilitating terminal erythroid maturation. To address this hypothesis, we generated novel HUDEP2 lines that OE HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb (Mbonye Proteomics 2015). Biotinylated 7SK pulldown confirmed that the Y271A mutation maintains the ability to bind the 7SK complex in erythroid cell extracts and RNA immunoprecipitation confirmed that the Y271A mutation increases the affinity of HEXIM1 for the 7SK complex in HUDEP2 cells. The Y271A mutation has significant functional consequences in erythroid cells. OE of wild type (WT) HEXIM1 in HUDEP2 cells resulted in enhanced proliferation in both expansion and maturation conditions, which was accompanied by increased cell and nuclear size, and a dramatic increase in the level of CD235a. Similar to our previously published HEXIM1 mutant with tyrosine to phenylalanine mutations at residues 271 and 274, the Y271A HEXIM1 mutation abrogated the enhanced proliferation seen with HEXIM1 OE in both expansion and maturation conditions. The Y271A mutation also rescued the larger cell and nuclear area associated with HEXIM1 OE, as well as the dramatic increase in the level of CD235a. Conversely, disruption of HEXIM1 via genome editing resulted in poor expansion and viability of HUDEP2 cells, which was rescued by expression of WT but not Y271A mutated HEXIM1, highlighting the importance of HEXIM1-pTEFb interactions for erythroid proliferation and survival. Further, OE of WT HEXIM1, but not the Y271A mutant, promoted erythroid gene expression while facilitating repression of genes that are normally silenced during terminal maturation, such as RPS19. In cells expressing WT HEXIM1 these gene expression changes were accompanied by increases in the global levels of ser2 and ser5 phosphorylated Pol II, as well as genome wide changes in their distribution. In contrast, the Y271A mutant decreased the global level of ser2 and ser5 pol II, consistent with its reduced ability to release pTEFb at target genes. Intriguingly, levels of H3K79me2, a histone mark reflective of active transcription through gene bodies, were decreased with OE of both WT and Y271A mutant HEXIM1, suggesting that the ability of HEXIM1 to promote transcriptional activation or repression is context dependent. Together, these data demonstrate a critical role for HEXIM1 and its interaction with pTEFb and the 7SK complex in the establishment of appropriate patterns of gene expression and chromatin architecture in maturing erythroblasts. No relevant conflicts of interest to declare.