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

红系终末成熟过程中RNA聚合酶II启动子近端暂停功能的研究

• 批准号: 10375479
• 负责人: LAURIE A. STEINER
• 金额: $ 33.88万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10375479
• 关键词: Address; Adult; Anemia; Basophils; Biological Assay; Cell Nucleus; ChIP-seq; Chromatin; Complex; Coupled; DNA Polymerase II; Data; Defect; Disease; Dysmyelopoietic Syndromes; Enhancers; Environment; Epigenetic Process; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; GATA1 gene; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Heterochromatin; Histone Acetylation; Histone Deacetylation; Histones; Human; Inherited; Investigation; Literature; Mass Spectrum Analysis; Modeling; Modification; Molecular; Nuclear; Phosphorylation; Physical condensation; Play; Post-Translational Protein Processing; Preparation; Process; Production; Proteins; Publishing; RNA; RNA Polymerase II; Regulation; Regulator Genes; Role; Small Nuclear Ribonucleoproteins; Suggestion; Tissues; Transcription Elongation; Transcription Initiation Site; Transcriptional Regulation; cell type; genome-wide; in vivo; insight; novel; prevent; progenitor; promoter; recruit; transcription factor

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.

末端红斑成熟涉及在核中基因表达的快速变化，而在核的背景下 正在逐步凝结生成。红斑成熟的每个阶段都与 一个不同的基因表达谱，但是与两者相关的表观遗传标记的分布 启动子和反射异染色质在红斑成熟的成功阶段相对静态。 相反，我们的初步数据表明，Hisstone标记与主动转录相关 延长（例如H3K36me3）在终末成熟期间发生巨大变化，表明红细胞 优先调节伸长水平的转录。 RNA聚合酶II暂停（pol II暂停）是 转录的高度调节机制，从而开始转录，但“停顿” 30-60bp 转录起始站点的下游。暂停是基因表达的关键检查点，因为Pol II不能 过渡到活跃的伸长，而不会被PTEFB磷酸化。 PTEFB可以与组织关联 包括GATA1在内的特定转录因子，以促进特定基因座的Pol II暂停释放，或者可以是 在7sk小核核糖核蛋白（SNRNP）复合物中被Hexim1隔离 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 是红斑成熟的关键调节剂，但是，Pol II暂停的机制在 成熟的红细胞知之甚少。支持Pol II暂停的核心作用 红细胞，质谱法证明了末端红细胞的成熟与减少有关 与主动转录伸长相关的多个Hisstone痕迹，再加上 标记的变化暗示了Pol II暂停的增加，而没有相关的异染色质增加。 CHIP-SEQ研究证实，H3K36me3的抽象减少与H3K36Me3的损失相关 富集在> 1600个位点。此外，Pol II暂停的中心驱动器Hexim1在红系中高度表达 与其他细胞类型相比，细胞的表达在整个RNA和蛋白质水平上都保持 末端红斑成熟。相反，PTEFB的表达和伸长水平也下降了 胜任的pol II。最后，Hexim1的诱导促进末端红斑成熟，并特别影响 在成熟过程中失去H3K36me3富集的基因的表达。一起我们的初步数据 支持我们的核心假设，即pol II暂停动态的转变越来越有利于“暂停” 状态是末端红细胞成熟的关键调节剂。在AIM1中，我们将描述Pol II的动态 暂停成熟的红细胞，我们将确定改变Pol II的后果暂停动力学 在末端红细胞成熟的多个方面。在AIM 2中，我们将确定特定机制 Pol II暂停在成熟的红细胞细胞中的特定局部建立并释放。这些研究将在一起 有助于重新定义我们概念化正常和无序的红细胞生成的范式。