The Biophysics of Developmentally Regulated Gene Transcription

发育调控基因转录的生物物理学

• 批准号: RGPIN-2017-04133
• 负责人: Mcghee, James
• 金额: $ 2.04万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=682848
• 关键词: Biophysics; Developmentally; Regulated; Gene; Transcription

Initiation of gene transcription is arguably the most influential control point in the development of a multicellular organism. In the simplest version of gene activation, a specific transcription factor binds to a short DNA sequence (6-12 bps) in the control region of a target gene and then somehow recruits the vast biochemical machinery of RNA Polymerase II and its auxiliary proteins, thereby initiating transcription. All of this occurs in an environment of “chromatin”, featuring possible cofactors, differentially modified histones and potentially positioned nucleosomes. We will focus on the all-important early step of gene activation in which a single controlling transcription factor interacts with its binding site in the regulatory region of a target gene. Our goal is to relate precise biophysical measurements made with purified components in vitro to quantitative transcriptional consequences measured in vivo.****For years, we have been analyzing and describing the development of a simple clonal cell lineage, the intestine of the small free-living nematode worm Caenorhabditis elegans. All the major transcription factors are now known but we will focus on ELT-2, the predominant transcription factor driving intestinal differentiation from the early embryo until death. We propose experiments to define three fundamental aspects of the interaction between ELT-2 and its binding sites inside an embryo.****Aim 1: ELT-2 binds preferentially to TGATAA DNA sites but different flanking sequences cause different binding affinities. Using advanced microscopic techniques, we will test if ELT-2 has the same spectrum of affinities binding to DNA in vitro and in vivo, i.e. inside the nucleus of a living embryo. We will also measure the level of free ELT-2 protein inside a living embryo. Both results are necessary to understand gene regulation at a quantitative biophysical level.****Aim 2: We have developed a tightly controlled experimental system in which single base pair variants of the ELT-2 binding site have significant transcriptional consequences inside the animal. We will first define the relation between ELT-2 affinity and transcript levels. and then extend this system to measure influences of developmental stage, chromatin proteins and chromatin modifications. We will ask the key question: do other target genes follow the same rules?****Aim 3: We will combine “low-tech” dissection of C. elegans intestines with state-of-the-art tagging of accessible DNA, thereby defining the position of nucleosomes in regulatory regions of intestine specific genes.****The aim of this research is to understand, at the fundamental level of binding energies, how critical decisions are made in embryonic development. Because ELT-2 is homologous to transcription factors GATA-4/5/6 driving endoderm development in vertebrates, the findings are likely to apply to development of intestines in all animals, including ourselves.***

基因转录的起始被认为是多细胞生物发育中最具影响力的控制点。在基因激活的最简单版本中，特定的转录因子与靶基因控制区的短DNA序列（6- 12bps）结合，然后以某种方式招募RNA聚合酶II及其辅助蛋白的庞大生化机制，从而启动转录。所有这些都发生在“染色质”的环境中，具有可能的辅因子，差异修饰的组蛋白和潜在的定位核小体。我们将重点关注基因激活的所有重要的早期步骤，其中单个控制转录因子与其在靶基因调控区域的结合位点相互作用。我们的目标是将用体外纯化组分进行的精确生物物理测量与体内测量的定量转录结果联系起来。****多年来，我们一直在分析和描述一个简单的克隆细胞系的发展，即小的自由生活的线虫线虫的肠道。所有主要的转录因子现在都是已知的，但我们将重点关注ELT-2，从早期胚胎到死亡驱动肠道分化的主要转录因子。我们建议通过实验来确定胚胎中ELT-2与其结合位点之间相互作用的三个基本方面。****目的1:ELT-2优先结合TGATAA DNA位点，但不同的侧翼序列导致不同的结合亲和力。使用先进的显微技术，我们将测试ELT-2在体外和体内（即在活胚胎的细胞核内）是否具有与DNA结合的相同亲和谱。我们还将测量活胚胎内游离ELT-2蛋白的水平。这两个结果都是在定量生物物理水平上理解基因调控的必要条件。****目标2：我们开发了一种严格控制的实验系统，在该系统中，ELT-2结合位点的单碱基对变体在动物体内具有显著的转录后果。我们将首先定义ELT-2亲和力和转录水平之间的关系。然后将该系统扩展到测量发育阶段、染色质蛋白和染色质修饰的影响。我们将提出一个关键问题：其他靶基因是否遵循同样的规则？****目标3：我们将结合秀丽隐杆线虫肠道的“低技术”解剖与最先进的可接近DNA标记，从而确定核小体在肠道特定基因调控区域的位置。****这项研究的目的是了解，在结合能的基本水平上，关键的决定是如何在胚胎发育中做出的。因为ELT-2与驱动脊椎动物内胚层发育的转录因子GATA-4/5/6是同源的，所以这些发现可能适用于所有动物的肠道发育，包括我们自己