Role of Negative Elongation Factor Complex in RNA polymerase II pausing and gene transcription in Heart

项目总结/摘要 充血性心力衰竭是美国和世界上发病率和死亡率的主要原因之一。 然而，尽管在研究和治疗发展方面取得了重大进展， 多年来，死亡率有所下降。这就需要重新研究管理的基本机制 心力衰竭的进展基因表达的适应性是人类免疫过程中最早的基本反应。 超载。我们已经证明了启动子近端RNA聚合酶II（pol II）的广泛调节作用 暂停在心脏基因转录。然而，控制和同步 释放暂停的pol II进行活性转录尚不清楚且相互矛盾，其对开发的贡献 心脏肥大和衰竭的原因还不清楚负延伸因子（Nelf），由五个亚基组成 （NelfA至NelfE）与pol II暂停有关，NelfA亚基被鉴定为重要组分 暂停。我们的初步数据显示，NelfA表达增加与心脏肥大，这是必要的， 以补偿这些心脏中基因表达的增加。相反，NelfA水平在衰竭的心脏中下降， 提示NelfA的下调可能导致失代偿和衰竭的进展。我们 全基因组测序数据显示NelfA在活性启动子上的广泛占据， 组成型表达的必需基因。有趣的是，染色质结合NelfA的免疫沉淀显示， 与染色质重塑和前mRNA加工蛋白的关联。在这项研究中，我们调查了 NelfA在pol II动力学、染色质重塑和基因表达中的作用及其对心脏疾病进展的影响 失败我们假设心肌肥厚过程中基因表达的适应是通过 - 暂停的pol II从必需基因启动子的清除速率的磷酸化依赖性增加，和 NelfA的从头募集和在诱导型启动子处暂停的复合物的组装。NelfA的缺失导致 中断暂停复合体，改变染色质重塑，低效的转录加工和抑制基因 导致心力衰竭的表达。我们提出了两个强大的具体目标来测试我们的假设-1。 研究NelfA依赖的pol II在心脏过程中基因转录暂停的调节机制， 肥厚2.研究NelfA缺失对心力衰竭基因表达和进展的影响， 条件NelfA-KO模型承受压力过载。

PROJECT SUMMARY/ABSTRACT Congestive heart failure is one of the leading cause of morbidity and mortality in the USA and the World. However, despite the major advancements in research and therapeutic developments, there has been no improvement in death rates over the years. This necessitates reinvestigating the basic mechanisms that govern the progression heart failure. Adaptation of gene expression is the earliest fundamental response during overload. We have shown the widespread regulatory influence of promoter-proximal RNA polymerase II (pol II) pausing on gene transcription in heart. However, the underlying mechanisms that control and synchronize the release of paused pol II for active transcription are unclear and conflicting, and its contribution to development of cardiac hypertrophy and failure still unknown. Negative elongation factors (Nelf), comprising of five subunits (NelfA to NelfE) has been implicated in pol II pausing, with NelfA subunit identified as an essential component for pausing. Our preliminary data shows increase in NelfA expression with cardiac hypertrophy, which is required for compensatory increase in gene expression in these hearts. Conversely, NelfA levels decline in failing hearts, suggesting downregulation of NelfA could be contributing to decompensation and progression of failure. Our genome-wide sequencing data shows widespread NelfA occupancy on active promoters including inducible and constitutively expressed essential genes. Interestingly, immunoprecipitation of chromatin bound NelfA shows association with chromatin remodelers and pre-mRNA processing proteins. In this study, we investigate the role of NelfA in pol II dynamics, chromatin remodeling and gene expression, and its impact on progression of heart failure. We have hypothesized that adaptation of gene expression during cardiac hypertrophy is achieved by phosphorylation dependent increase in the rate of clearance of paused pol II from essential gene promoters, and de novo recruitment of NelfA and assembly of paused complex at inducible promoters. Loss of NelfA results in disrupted paused complex, altered chromatin remodeling, inefficient transcript processing and inhibited gene expression that precipitates heart failure. We have proposed two robust specific aims to test our hypothesis – 1. To examine the mechanisms regulating NelfA -dependent pol II pausing in gene transcription during cardiac hypertrophy. 2. To investigate the effects of loss of NelfA on gene expression and progression of heart failure in conditional NelfA-KO model subjected to pressure overload.