Coordinate Gene Regulation in Animal Cells

描述（由申请人提供）：真核生物成熟mRNA的调控生产需要高度协调的分子相互作用和涉及数百个多肽参与的生化过程。了解这些潜在的过程对于创建干预由突变、表观遗传异常或感染因子引起的异常调节的新策略非常重要。热休克（HS）基因是一组高度调控的基因，特别适合研究诱导mRNA产生的基本特征。在这里，成熟的和新开发的技术将被用来辨别启动子结构和调节机制的关键特征。热休克基因和许多重要的高度调控基因的启动子区域的一个关键特征是启动子近端RNA聚合酶II暂停（暂停Pol II）。在本应用的目标1中，将使用我们新开发的Global nuclear Run-On和大规模平行测序协议（GRO- seq）的一种变体来研究暂停Pol II的建立和性质，该协议旨在提供果蝇细胞全基因组范围内暂停Pol II的近核苷酸分辨率图谱。结合生物信息学方法对所有暂停的精确定位将极大地有助于确定暂停的Pol II调节中使用的一般元件和因素。DNA元素的作用及其相互之间的空间关系将通过对现有模式基因（如Hsp70）和GRO-seq分析发现的新基因的靶向诱变来测试。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对暂停rna的敏感和高分辨率生化分析来定义暂停Pol II的重要机制和动态特性。在Aim 2中，我们通过对与主调控因子HSF相互作用的因子进行全面的生化搜索，并分析了我们从定向和全基因组筛选中获得的影响Hsp70表达的因素，研究了激活启动子-近端暂停Pol II的机制。现有的和新发现的转录因子将被检查，以确定何时何地参与体内的激活过程，通过ChIP测定和双光子显微镜来实时跟踪特定激活位点上因子的招募和动态。在Aim 3中，我们研究了导致核小体在整个活化位点上快速大规模丢失的机制。在HS中，核小体以转录无关的方式从热休克位点迅速丢失。核小体损失的区域延伸到基因之外，直至但不超过绝缘体位点、scs和scs’。核小体的全位点损失取决于HSF和PARP。本研究将探讨HSF和PARP在基因激活和核小体丢失过程中的空间关系，检测HSF和PARP之间的相互作用，并确定scs和scs绝缘体区域核小体丢失的关键屏障。

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.