Investigating how transcription factors cooperate and overcome the enhancer nucleosome barrier during embryonic patterning

研究胚胎模式形成过程中转录因子如何合作并克服增强子核小体屏障

• 批准号: 10584476
• 负责人: Kaelan Joseph Brennan
• 金额: $ 3.17万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584476
• 关键词: ATAC-seq; Address; Affect; Automobile Driving; Binding; Cell Reprogramming; Cells; Chemicals; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Sequence; DNA-Binding Proteins; Data; Development; Diabetes Mellitus; Disease; Dorsal; Drosophila genus; Drosophila melanogaster; Embryo; Embryonic Development; Engineering; Enhancers; Excision; Exposure to; Face; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genome; Genomic approach; Genomics; Histones; Human body; Malignant Neoplasms; Maps; Mediating; Modeling; Mutation; Nucleosomes; Obesity; Pattern; Pattern Formation; Positioning Attribute; Proteins; Regulation; Reporter Genes; Resolution; System; Techniques; Testing; Time; Tissues; Transgenic Organisms; Variant; Work; base; cell type; convolutional neural network; deep learning model; developmental disease; experimental study; genome-wide; genome-wide analysis; human disease; in vivo; insight; mutant; next generation sequencing; predictive modeling; prevent; programs; spatiotemporal; temporal measurement; transcription factor

Project Summary This misregulation of gene expression underlies several human diseases, including many cancers, diabetes, obesity, and multiple developmental disorders. Genome-wide studies and next-generation sequencing have revealed that sequence variants in enhancers, cis-regulatory DNA sequences that control spaciotemporal gene expression programs, contribute to the development of these diseases. These mutations often affect enhancer activity, which must be tightly controlled since enhancers drive tissue and cell-type specific gene expression patterns. One way that enhancer activity is controlled is through the regulation of enhancer accessibility by the nucleosome: the structural unit of chromatin comprised of 147 bp of DNA and a histone octamer. Enhancers are characterized by an intrinsically strong nucleosome barrier that prevents the binding of transcription factors (TFs), the proteins that activate enhancers, until the proper context for activation is reached, at which point TFs must overcome the nucleosome barrier and bind to the DNA. While nucleosome depletion is a key early step in enhancer activation, we do not yet understand how the nucleosome barrier is overcome and how enhancers are made accessible for gene activation, despite accessibility being a major regulator of enhancer activity. Current models suggest that specialized TFs called pioneer factors can access their motifs in the presence of nucleosomes and foment nucleosome depletion through cooperativity with additional TFs. Even still, how pioneer and non-pioneer TFs cooperate to generate chromatin accessibility at enhancers is not yet known. Furthermore, how pioneer factors perturb the nucleosomal landscape to facilitate chromatin accessibility and cooperative TF binding is unclear. This study seeks to identify how TFs overcome the nucleosome barrier at enhancers using high-resolution experimental and computational genomics techniques to map TF binding, chromatin accessibility, and nucleosome positioning. Aim 1 will characterize how pioneer and non-pioneer TFs cooperate for binding to the DNA and for establishing chromatin accessibility. This aim will combine high-resolution TF binding (ChIP- nexus) and temporally resolved chromatin accessibility (time-course ATAC-seq) information with deep learning models (BPNet) that will reveal the sequences and sequence constraints that are important for and predictive of TF cooperativity. Aim 2 will profile genome-wide nucleosome positional changes over developmental time at unprecedented resolution, using a chemical mapping of nucleosome centers approach. This aim will uncover how the nucleosome state at enhancers is altered over time to generate accessibility and how nucleosomes are positioned with respect to the underlying regulatory DNA sequences. Taken together, these aims will illuminate how TFs pioneer the chromatin landscape for enhancer activation, thereby deepening the field’s understanding of the mechanisms of gene regulation and how misregulation contributes to human disease.

项目摘要 这种基因表达的失调是几种人类疾病的基础，包括许多癌症、糖尿病、 肥胖和多发性发育障碍。全基因组研究和下一代测序 揭示了增强子中的序列变异，即控制时空基因的顺式调控DNA序列 表达计划，有助于这些疾病的发展。这些突变经常影响增强子 活性，必须严格控制，因为增强剂推动组织和细胞类型的特定基因表达 模式。控制增强子活性的一种方式是通过 核小体：染色质的结构单位，由147bp的DNA和一个组蛋白八聚体组成。增强剂 以阻止转录因子结合的内在强大的核小体屏障为特征 (TFS)，激活增强子的蛋白质，直到达到适当的激活环境，在这一点上 必须克服核小体障碍并与DNA结合。虽然核小体耗尽是早期的一个关键步骤 增强子激活，我们还不知道核小体障碍是如何克服的，以及增强剂是如何 尽管可及性是增强子活性的主要调节因素，但基因激活是可获得的。 目前的模型表明，被称为先锋因子的特殊因子可以在存在的情况下获得它们的主题 核小体，并通过与额外的转录因子的协同作用而加剧核小体的耗竭。即使是这样，如何 先驱者和非先驱者TF在增强剂中合作产生染色质可及性尚不清楚。 此外，先驱因素如何扰乱核小体的格局，以促进染色质的可及性和 协同转铁蛋白结合尚不清楚。 这项研究试图确定转录因子如何利用高分辨率克服增强剂中的核小体障碍。 实验和计算基因组学技术以定位转铁蛋白结合、染色质可及性和 核小体定位。目标1将描述先锋和非先锋TF如何合作以约束 DNA和建立染色质的可及性。这一目标将结合高分辨率的TF结合(芯片- Nexus)和具有深度学习的时间分辨染色质可获得性(时程ATAC-SEQ)信息 将揭示序列和序列约束的模型(BPNet)，这些序列和序列约束对 转移因子的协作性。AIM 2将在以下位置描述全基因组核小体位置随发育时间的变化 前所未有的分辨率，使用了一种化学测绘核小体中心的方法。这一目标将揭开 增强剂上的核小体状态如何随时间改变以产生可及性，以及核小体如何 相对于潜在的调控DNA序列进行定位。总而言之，这些目标将 阐明TF如何开创染色质领域的先河，促进增强子的激活，从而深化该领域的 了解基因调控的机制以及调控不当如何导致人类疾病。