Terminal erythroid maturation involves rapid changes in gene expression in the context of a nucleus that is progressively condensing in preparation for enucleation. Each stage of erythroid maturation is associated with a distinct gene expression profile, however the distribution of epigenetic marks associated with both active promoters and repressed heterochromatin is relatively static between successive stages of erythroid maturation. In contrast, our preliminary data demonstrates that histone marks associated with active transcriptional elongation, such as H3K36me3, change dramatically during terminal maturation, suggesting erythroblasts preferentially regulate transcription at the level of elongation. RNA Polymerase II pausing (Pol II pausing) is a highly regulated mechanism of transcriptional regulation whereby transcription is initiated, but ?pauses? 30-60bp downstream of the transcription start site. Pausing is a critical checkpoint in gene expression, as pol II cannot transition into active elongation without being phosphorylated by pTEFb. pTEFb can associate with tissue specific transcription factors, including GATA1, to facilitate pol II pause release at specific loci, or it can be sequestered by Hexim1 in the 7sk small nuclear ribonucleoprotein (snRNP) complex rendering it in active and unable to facilitate Pol II release. Both our preliminary data and the published literature suggest that Pol II pausing is a critical regulator of erythroid maturation, however the mechanisms by which Pol II pausing is regulated in maturing erythroblasts are poorly understood. Supporting a central role for Pol II pausing in maturing erythroblasts, mass spectrometry demonstrates that terminal erythroid maturation is associated with a decrease in the abundance of multiple histone marks associated with active transcriptional elongation, coupled with changes in marks suggestive of increased Pol II pausing, without an associated increase in heterochromatin. ChIP-seq studies confirm that the decrease in abundance of H3K36me3 is correlated with loss of H3K36me3 enrichment at >1600 loci. In addition, Hexim1, a central driver of pol II pausing, is highly expressed in erythroid cells compared to other cell types and its expression is maintained at both the RNA and protein level throughout terminal erythroid maturation. In contrast, the expression of pTEFb declines, as does the level of elongation competent Pol II. Lastly, induction of hexim1 promotes terminal erythroid maturation, and specifically impacts the expression of genes that lose enrichment for H3K36me3 during maturation. Together our preliminary data support our central hypothesis that a shift in Pol II pause dynamics that increasingly favors the ?paused? state is a critical regulator of terminal erythroid maturation.
In aim1, we will delineate the dynamics of Pol II pausing in maturating erythroblasts and we will determine the consequences of altering Pol II pausing dynamics on multiple facets of terminal erythroid maturation.
In aim 2, we will determine the specific mechanisms by which Pol II pausing is established and released at specific loci in maturing erythroid cells. Together, these studies will help to redefine the paradigm with which we conceptualize both normal and disordered erythropoiesis.
The average adult generates >2 million red blood cells per second. The process of generating a mature red blood cell from a committed progenitor is complex, and defects in this process result in anemia. The goal of this proposal is to understand how a specialized type of gene regulation, RNA polymerase II proximal promoter pausing, regulates red blood cell production.
