Erythropoiesis is a finely orchestrated process that involves generating a ~2.5 million red blood cells per second to maintain homeostasis and prevent anemia. RNA Polymerase II (RNAPII) pausing is a highly regulated and fundamental mechanism of transcriptional regulation, whereby transcription is initiated, but pauses 30-60 bp downstream of the transcription start site. Release of paused RNAPII into active elongation requires phosphorylation of RNAPII and associated inhibitory factors by positive transcription elongation factor beta (pTEFb). Our group has shown that regulation of RNAPII activity is an essential determinant of erythroid cell function, with central roles in the regulation of gene expression and cell cycle progression. HEXIM1 is a key regulator of pTEFb activity that can enforce pausing or facilitate pause release, depending on genomic context. HEXIM1 is essential for erythropoiesis, and promotes erythroid proliferation and the expression of GATA1-target genes. Despite its importance, fundamental questions remain regarding the mechanisms by which HEXIM1 regulates RNAPII activity and promotes erythroid gene expression. HEXIM1 is classically thought of as a transcriptional repressor, which enforces RNAPII pausing by sequestering pTEFb in the 7SK complex. Data from our group and others suggests the HEXIM1-pTEFb-7sk complex can be targeted to specific genes, where it is available for “onsite” release of pTEFb, facilitating pause release. In Aim 1, we will address the hypothesis that delivery of pTEFb to specific genes by HEXIM1 and the 7SK Complex is essential for both steady state and stress erythropoiesis. RNAPII pausing is regulated by cell type- and maturation stage-specific transcription factors and signaling pathways. Early erythropoiesis is comprised of the differentiation of multipotent progenitors to erythroid progenitors, while terminal maturation consists of the maturation of proerythroblasts through several intermediates into orthochromatic erythroblasts that ultimately enucleate. In contrast to terminally maturing cells, erythroid progenitors have the ability to undergo self-renewal divisions, and to expand in response to anemic stress. HEXIM1 is expressed in erythroid progenitors and upregulated in terminally maturing cells. Other key regulators of pTEFb, including Bromodomain Protein 4 (BRD4), and the Mediator Complex are highly expressed in erythroid progenitors and down regulated during terminal maturation. Erythroid progenitors also have higher levels of RNAPII than terminally maturing cells. These data suggest that the transcriptional environment of erythroid progenitors is distinct from that of terminally maturing cells. In Aim2, we will address the hypothesis that RNAPII activity is regulated in a maturation stage-specific manner. The proposed studies will provide novel insights into the mechanisms that govern normal and perturbed erythropoiesis, and have the potential to identify novel therapeutic targets for inher...