Molecular Mechanisms of Promoter-Terminator Gene Looping

 描述（由申请人提供）：越来越多的证据表明，高阶基因组结构影响转录并介导不同DNA元件之间的关键共转录串扰。近年来，多种类型的染色质环（如启动子-终止子、增强子-启动子和染色体间环）在发育过程中和癌细胞中的基因调控中的功能意义已得到证实。来自转录起始和mRNA 3 '端切割和多聚腺苷酸化（poly（A））机制的必需因子已显示形成复合物以赋予启动子-终止子基因环。一般转录因子TFIIB介导涉及Ssu 72（pol II C-末端结构域磷酸酶）和Rna 15（切割因子IA复合物的poly（A）RNA结合亚基）的关键相互作用网络。基因成环是酵母中普遍存在的现象，也已在哺乳动物基因、植物基因和人类免疫缺陷病毒前病毒中发现。此外，基因环已连接到一些基本的细胞过程，其中包括转录重新启动，转录记忆的维持，和转录方向性的控制。 在本研究中，我们将解决以下关键问题：（1）TFIIB和以TFIIB为中心的基因环复合物如何调节转录（目的1），（2）pol II转录偶联基因的结构和生化基础是什么， 环的建立和维持（目标2），（3）基因环依赖性pol II再起始（目标3）的机制和生理后果是什么？我们广泛的假设是，由核因子和非编码RNA形成的独特的大分子复合物介导基因循环，并创造独特的局部染色质环境来控制pol II转录。具体而言，我们将通过以下方式来验证这一假设：（1）在生物化学和结构上绘制以TFIIB为中心的基因环复合物的结合界面，并将其直接与体内基因环形成过程相联系;（2）揭示转录过程中TFIIB依赖性基因环形成的途径，并鉴定其他核因子（例如，粘着蛋白、凝聚蛋白、核孔复合物和TFIIIC），以及（3）用包括竞争ChIP、核锚离系统，和化学诱导的人工基因环系统。总的来说，建立在我们的初步结果，这些研究将提供基因环及其介导的共转录串扰的分子机制。此外，我们将通过识别对这一过程至关重要的其他核因子将基因循环与其他细胞途径联系起来。最后，我们的研究可能会深入了解与发育和人类疾病有关的所有类型的转录依赖性染色质环的共同原理，从而为治疗提供新的靶点。

 DESCRIPTION (provided by applicant): Accumulating evidence has indicated that high-order genome structures influence transcription and mediate critical co-transcriptional crosstalk among distinct DNA elements. The functional significance of a variety of types of chromatin loops (e.g. promoter-terminator, enhancer-promoter, and interchromosomal) in gene regulation during development and in cancer cells has been demonstrated recently. Essential factors from the transcription initiation and mRNA 3'-end cleavage and polyadenylation (poly(A)) machineries have been shown to form complexes to confer promoter-terminator gene looping. The general transcription factor TFIIB mediates a critical interaction network involving Ssu72 (a pol II C-terminal domain phosphatase) and Rna15 (the poly(A) RNA-binding subunit of the cleavage factor IA complex). Gene looping is a widespread phenomenon in yeast and has also been identified for mammalian genes, plant genes, and human immunodeficiency virus provirus. In addition, gene looping has been connected to a number of fundamental cellular processes, which include transcription reinitiation, maintenance of transcriptional memory, and control of transcriptional directionality. In the proposed research, we will address the following key questions: (1) how do TFIIB and TFIIB- centered gene looping complexes regulate transcription (Aim 1), (2) what is the structural and biochemical basis for the pol II transcription-coupled gene loop establishment and maintenance (Aim 2), and (3) what are the mechanisms and physiological consequences of gene looping-dependent pol II reinitiation (Aim 3)? Our broad hypothesis is that unique macromolecular complexes formed by nuclear factors and non-coding RNAs mediate gene looping and create unique local chromatin environments to control pol II transcription. Specifically, we will test this hypothesis by (1) biochemically and structurally mapping binding interfaces of the TFIIB-centered gene looping complexes and connect them to the gene looping process directly in vivo, (2) revealing the pathways of TFIIB-dependent gene loop formation during the course of transcription and identifying additional nuclear factors (e.g. cohesin, condensin, nuclear pore complexes, and TFIIIC) required for gene looping, and (3) determining pol II and transcription factor dynamics on chromatin governed by gene loops with tools including the competition ChIP, the nuclear anchor-away system, and the chemically induced artificial gene looping system. Collectively, built upon our preliminary results these studies will provide the molecular mechanisms of gene looping and its mediated co-transcriptional crosstalk. In addition, we will link gene looping to other cellular pathways by identifying additional nuclear factors critical for this process. Finally, our studies may offer insights into the common principles of all types of transcription-dependent chromatin loops implicated in development and human disease and hence suggest novel targets for therapeutics.