Coordinate Gene Regulation in Animal Cells

描述(申请人提供)：真核生物中成熟信使核糖核酸的调控生产需要高度协调的分子相互作用和生化过程，涉及数百个多肽的参与。了解这些潜在的过程对于创造干预由突变、表观遗传异常或感染性病原体引起的异常调控的新策略非常重要。热休克(HS)基因是一组高度调控的基因，特别适合于研究可诱导mRNA产生的基本特征。在这里，成熟的和新开发的技术都将被用来识别启动子结构和调控机制的关键特征。热休克基因和许多重要的高调控基因启动子区域的一个重要特征是启动子-近端RNA聚合酶II暂停(Pol II暂停)。在本申请的目标1中，将使用我们新开发的全球核连续和大规模并行测序协议(GRO-SEQ)的变体来研究暂停的POL II的建立和性质，该协议旨在提供暂停的POL II在基因组范围内的基因组范围内的近核苷酸分辨率图谱。所有暂停的精确定位，加上生物信息学方法，应极大地有助于确定暂停的POL II规章中使用的一般要素和因素。DNA元素的作用及其相互之间的空间关系将通过对现有的模型基因(如Hsp70)和Gro-seq分析发现的新基因进行定向突变来测试。我们还将使用我们最新开发的活细胞成像方法和经典的脉冲标记实验，结合对暂停的RNA进行灵敏和高分辨率的生化分析，来定义暂停的POL II的重要机制和动态特性。在目标2中，我们通过对与主调控因子HSF相互作用的因素进行全面的生化搜索，以及从定向筛选和全基因组筛选获得的影响Hsp70表达的因素的分析，来研究激活启动子-近端暂停的POL II的机制。现有的和新发现的转录因子将通过芯片分析和双光子显微镜来确定它们在体内参与激活过程的时间和地点，以及实时跟踪因子在特定激活位置的招募和动态。在目标3中，我们研究了在整个激活位点上导致核小体快速大规模丢失的机制。在HS中，核小体以一种转录无关的方式从热休克基因座迅速丢失。核小体丢失的区域延伸到基因之外，直到但不超过位点绝缘子，SCS和SCS‘。这种全区域的核小体丢失取决于HSF和PARP。这些分析将探索HSF和PARP在基因激活和核小体丢失过程中的空间关系，测试HSF和PARP之间的相互作用，并确定SCS和SCS绝缘区中防止核小体丢失的关键屏障。 与公共健康相关：生物体的发育、健康和生存依赖于大量的细胞内和细胞外信号，这些信号产生高度协调和调控的基因表达，其中大部分调控发生在转录水平。这项提案中的一些方法和正在开发的技术正在为活细胞复杂但相关的环境中的这些分子机制提供直接的见解，而另一些方法则提供了前所未有的关于启动子和基因结构和表达的全基因组视角。这些信息将为在基因调控和表达水平上了解正常和疾病状态提供必要的背景，并为设计干预与癌症相关基因的异常表达或因转录因子功能突变或中断而引起的疾病的策略提供必要的背景。

DESCRIPTION (provided by applicant): The regulated production of a mature mRNA in eukaryotes requires highly coordinated molecular interactions and biochemical processes that involve the participation of hundreds of polypeptides. Understanding these underlying processes is important in creating novel strategies for intervening in abnormal regulation caused by mutations, epigenetic abnormalities, or infectious agents. The heat shock (HS) genes are a highly regulated set of genes particularly well suited to investigate fundamental features of inducible mRNA production. Here, both well-established and newly developed technologies will be used to discern critical features of promoter architecture and mechanisms of regulation. A critical feature of the promoter regions of heat shock genes and many important highly regulated genes is promoter-proximal RNA Polymerase II pausing (Paused Pol II). In Aim 1 of this application, the establishment and properties of Paused Pol II will be investigated using a variation of our newly developed Global nuclear Run-On and massively-parallel sequencing protocol (GRO- seq) that is designed to provide near-nucleotide resolution mapping of Paused Pol II on a genome-wide scale in Drosophila cells. The precise positioning of all pauses coupled with bioinformatic approaches should greatly aid the identification of general elements and factors used in Paused Pol II regulation. The role of DNA elements and their spatial relationships to each other will be tested using targeted mutagenesis of existing model genes like Hsp70, and new genes uncovered by GRO-seq analyses. We will also define important mechanistic and dynamic properties of Paused Pol II using both our newly developed live cell imaging method and classical pulse-labeling experiments coupled with sensitive and high-resolution biochemical assays of paused RNAs. In Aim 2, we investigate the mechanism of activating promoter-proximal paused Pol II by performing a comprehensive biochemical search for factors that interact with master regulator, HSF, as well as analysis of factors affecting Hsp70 expression that we obtained from directed and genome-wide screens. Existing and newly identified transcription factors will be examined to determine when and where they participate in the activation process in vivo by ChIP assays as well as two-photon microscopy to track in real time the recruitment and dynamics of factors at specific activated loci. In Aim 3, we investigate the mechanisms that bring about rapid large scale loss of nucleosomes over an entire activated locus. Upon HS, nucleosomes are rapidly lost from heat shock loci in a transcription-independent manner. The region of nucleosome loss extends beyond the gene and up to, but not beyond, the locus insulators, scs and scs'. This locus-wide loss of nucleosomes depends on HSF and PARP. The proposed analyses will probe the spatial relationship of HSF and PARP during gene activation and nucleosome loss, testing for interactions between HSF and PARP, and identifying the critical barrier to nucleosome loss in the scs and scs' insulator regions. PUBLIC HEALTH RELEVANCE: The development, health, and viability of an organism depend on a plethora of intra- and extracellular signals that produce a highly orchestrated and regulated gene expression, with much of this regulation occurring at the level of transcription. Some of the approaches in this proposal and the technology being developed are providing direct insights to these molecular mechanisms in the complex but relevant milieu of living cells, while others provide unprecedented genome-wide views of promoter and gene architecture and expression. This information will provide the necessary background for understanding normal and disease states at the level of gene regulation and expression, and for designing strategies for intervening with abnormal expression of genes associated with cancer or disorders originating from mutation or disruption of transcription factor functions.