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

项目摘要/摘要 充血性心力衰竭是美国和世界范围内发病率和死亡率的主要原因之一。 然而，尽管在研究和治疗开发方面取得了重大进展，但没有 多年来死亡率的提高。这就需要重新研究治理的基本机制。 进行性心力衰竭。基因表达的适应是最早的基本反应 超载。我们已经证明了启动子-近端rna聚合酶II(PolII)的广泛调控作用。 暂停心脏中的基因转录。然而，控制和同步 暂停的polII的释放以激活转录是不清楚和相互矛盾的，以及它对发育的贡献 心肌肥大和衰竭的原因尚不清楚。负伸长因子(NELF)，由五个亚基组成 (NelfA到NelfE)与PolII暂停有关，NelfA亚基被认为是一个重要组成部分 因为我暂停了。我们的初步数据显示，随着心肌肥厚，NelfA的表达增加，这是必需的 在这些心脏中基因表达的补偿性增加。相反，在心脏衰竭时，NelfA水平会下降， 提示NelfA的下调可能导致失代偿和衰竭的进展。我们的 全基因组测序数据显示NelfA广泛存在于活性启动子上，包括可诱导的和 结构性地表达必要的基因。有趣的是，染色质结合的NelfA的免疫沉淀显示 与染色质重构体和前信使核糖核酸加工蛋白的关系。在这项研究中，我们调查了 NelfA在PolII动力学、染色质重塑和基因表达中的作用及其对心脏进展的影响 失败了。我们假设在心肌肥厚过程中基因表达的适应是通过 依赖于磷酸化的暂停的PolII从必需基因启动子中清除的速度增加，以及 NelfA的从头招募和在可诱导启动子上暂停的复合体的组装。NelfA的丢失导致 打乱暂停的复合体，改变染色质重塑，低效转录处理和抑制基因 导致心力衰竭的表情。我们提出了两个稳健的具体目标来检验我们的假设-1。 心脏组织中NelfA依赖的PolII基因转录暂停的调节机制 肥大。2.探讨NelfA缺失对心力衰竭大鼠基因表达及心力衰竭进展的影响。 压力过载下的条件NelfA-KO模型。

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.