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，并在不同的DNA元素之间调节关键的共转录串扰。各种类型的染色质环(如启动子-终止子、增强子-启动子和染色体间)在发育过程中和癌细胞中的基因调控中的功能意义最近被证明。转录起始、mRNA3‘端切割和多聚腺苷化(PolyA)机制中的基本因子已被证明形成复合体，从而实现启动子-终止子基因环。通用转录因子TFIIB介导了一个关键的相互作用网络，涉及Ssu72(PolII C末端结构域磷酸酶)和Rna 15(切割因子IA复合体的聚(A)RNA结合亚单位)。基因环在酵母中是一种普遍存在的现象，在哺乳动物基因、植物基因和人类免疫缺陷病毒前病毒中也被发现。此外，基因环与许多基本的细胞过程有关，包括转录重新启动、转录记忆的维持和转录方向性的控制。在拟议的研究中，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体如何调控转录(目标1)；(2)polII转录偶联基因的结构和生化基础是什么 环的建立和维护(目标2)，以及(3)依赖基因环的POLII重新启动的机制和生理后果是什么(目标3)？我们的广泛假设是，由核因子和非编码RNA形成的独特的大分子复合体介导了基因环，并创造了独特的局部染色质环境来控制PolII的转录。具体地说，我们将通过以下方式检验这一假说：(1)以TFIIB为中心的基因环复合体的结合界面的生化和结构图谱，并将它们直接连接到体内的基因环过程；(2)揭示转录过程中依赖TFIIB的基因环形成的途径，并识别基因环所需的额外核因子(例如粘附素、凝集素、核孔复合体和TFIIIC)；以及(3)利用竞争芯片、核锚定系统和化学诱导的人工基因环系统等工具，确定基因环控制的染色质上的PolII和转录因子动力学。总而言之，基于我们的初步结果，这些研究将提供基因循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，将基因环路与其他细胞途径联系起来。最后，我们的研究可能为所有类型的转录依赖染色质环在发育和人类疾病中的共同原理提供见解，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.