Coordinate Gene Regulation in Animal Cells

描述（由申请人提供）：真核生物中成熟mRNA的调节产生需要高度协调的分子相互作用和涉及数百种多肽参与的生化过程。了解这些潜在的过程是非常重要的，在创造新的策略，干预由突变，表观遗传异常，或感染性病原体引起的异常调节。热休克（HS）基因是一组高度调控的基因，特别适合研究诱导型mRNA产生的基本特征。在这里，既有成熟的和新开发的技术将用于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度调节基因的启动子区的一个关键特征是启动子近端RNA聚合酶II暂停（Paused Pol II）。在本申请的目的1中，将使用我们新开发的全局核Run-On和并行测序方案（GRO-seq）的变体来研究Paused Pol II的建立和性质，所述方案被设计为在果蝇细胞中在全基因组规模上提供Paused Pol II的近核苷酸分辨率作图。所有暂停的精确定位加上生物信息学方法，应大大有助于识别暂停Pol II调节中使用的一般元素和因素。DNA元件的作用和它们彼此之间的空间关系将使用现有模型基因如Hsp 70的靶向诱变和通过GRO-seq分析发现的新基因来测试。我们还将使用我们新开发的活细胞成像方法和经典的脉冲标记实验以及暂停RNA的灵敏和高分辨率生化测定来定义暂停Pol II的重要机制和动态特性。在目的2中，我们通过对与主调节因子HSF相互作用的因子进行全面的生化搜索，以及对我们从定向和全基因组筛选中获得的影响Hsp 70表达的因子进行分析，来研究激活启动子近端暂停的Pol II的机制。将检查现有和新鉴定的转录因子，以确定它们何时何地参与体内激活过程，方法是通过ChIP测定以及双光子显微镜以真实的时间跟踪特定激活位点因子的募集和动力学。在目标3中，我们研究了在整个激活位点上引起核小体快速大规模损失的机制。在HS时，核小体以不依赖于转录的方式从热休克位点迅速丢失。核小体丢失的区域延伸到基因之外，直到但不超过基因座绝缘体，scs和scs '。核小体的这种基因座范围的丢失取决于HSF和PARP。所提出的分析将探测基因激活和核小体损失期间HSF和PARP的空间关系，测试HSF和PARP之间的相互作用，并确定scs和scs绝缘子区域中核小体损失的关键障碍。 公共卫生相关性：生物体的发育、健康和生存能力取决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节的基因表达，其中大部分调节发生在转录水平。该提案中的一些方法和正在开发的技术为活细胞复杂但相关的环境中的这些分子机制提供了直接的见解，而其他方法则提供了前所未有的启动子和基因结构和表达的全基因组视图。这些信息将提供必要的背景，了解正常和疾病状态的基因调控和表达的水平，并为设计干预与癌症或源于突变或转录因子功能破坏的疾病相关的基因的异常表达的策略。

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.