Investigation into the function of RNA polymerase II promoter proximal pausing during terminal erythroid maturation

终末红细胞成熟涉及在细胞核中基因表达的快速变化， 正在逐渐浓缩为摘除做准备红细胞成熟的每个阶段都与 一个独特的基因表达谱，然而，与两个活性相关的表观遗传标记的分布 启动子和抑制的异染色质在红细胞成熟的连续阶段之间是相对静止的。 相反，我们的初步数据表明，组蛋白标记与活性转录相关， 延伸，如H3 K36 me 3，在终末成熟过程中发生显著变化，表明成红细胞 优先在延伸水平调节转录。RNA聚合酶II暂停（Pol II暂停）是一种 一种高度调节的转录调节机制，转录启动，但“暂停”30- 60 bp 转录起始位点的下游。暂停是基因表达的关键检查点，因为pol II不能 在不被pTEFb磷酸化的情况下转变成活性延伸。pTEFb可与组织结合 特异性转录因子，包括GATA 1，以促进pol II在特定位点的暂停释放，或者它可以是 被Hexim 1隔离在7sk小核核糖核蛋白（snRNP）复合物中，使其处于活性状态， 无法促进Pol II释放。我们的初步数据和已发表的文献都表明，Pol II暂停 是红系成熟的关键调节因子，然而，在红系成熟中调节Pol II暂停的机制是不确定的。 成熟的成红细胞知之甚少。支持Pol II暂停在成熟过程中发挥核心作用 质谱分析表明，终末红系成熟与成红细胞减少有关。 与活跃的转录延伸相关的多个组蛋白标记的丰度， 标志物的变化提示Pol II暂停增加，而异染色质没有相关增加。 ChIP-seq研究证实，H3 K36 me 3丰度的降低与H3 K36 me 3的丢失相关 富集在>1600个位点。此外，Hexim 1是pol II暂停的中心驱动因子，在红系细胞中高度表达。 与其他细胞类型相比，它的表达在整个过程中保持在RNA和蛋白质水平 红系终末成熟与此相反，pTEFb的表达下降，延长的水平也是如此。 有能力的Pol II。最后，hexim 1的诱导促进终末红系成熟，并且特别影响 在成熟过程中丧失对H3 K36 me 3富集的基因的表达。结合我们的初步数据 支持我们的中心假设，即Pol II暂停动态的转变越来越有利于“暂停” 状态是终末红细胞成熟的关键调节因子。在aim 1中，我们将描述Pol II的动态 我们将确定改变Pol II暂停动力学的后果 在红细胞成熟的多个方面。在目标2中，我们将确定具体机制， Pol II暂停在成熟红系细胞的特定位点建立和释放。这些研究将 帮助我们重新定义正常和异常红细胞生成的概念。

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.