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、 肥胖和多种发育障碍。全基因组研究和下一代测序已经 揭示了控制时空基因的增强子、顺式调节DNA序列中的序列变体 表达程序，有助于这些疾病的发展。这些突变通常会影响增强子 活性，必须严格控制，因为增强子驱动组织和细胞类型特异性基因表达 模式.控制增强子活性的一种方式是通过调节增强子的可及性， 核小体：染色质的结构单位，由147 bp的DNA和组蛋白八聚体组成。增强剂 其特征在于固有的强核小体屏障，其阻止转录因子的结合 (TFs)激活增强子的蛋白质，直到达到激活的适当环境，此时TF 必须克服核小体屏障与DNA结合。虽然核小体消耗是一个关键的早期步骤， 增强子激活，我们还不知道如何克服核小体障碍，以及增强子如何激活核小体。 尽管可接近性是增强子活性的主要调节因子，但可接近性对于基因激活是可接近的。 目前的模型表明，被称为先锋因子的特化TF可以在以下情况下访问它们的基序： 核小体并通过与另外的TF的协同性引起核小体消耗。即便如此， 先驱和非先驱TF合作在增强子处产生染色质可及性尚不清楚。 此外，先锋因子如何扰乱核小体景观，以促进染色质的可及性， 协同TF结合尚不清楚。 本研究旨在利用高分辨率的荧光显微镜， 实验和计算基因组学技术，以映射TF结合，染色质可及性， 核小体定位目标1将说明先驱和非先驱工作队如何合作， DNA并用于建立染色质可及性。这一目标将结合联合收割机高分辨率TF结合（ChIP- nexus）和时间分辨的染色质可及性（时程ATAC-seq）信息 模型（BPNet），将揭示序列和序列约束，这是重要的和预测 TF协同性Aim 2将描绘全基因组核小体在发育过程中的位置变化， 前所未有的分辨率，使用核小体中心的化学作图方法。这一目标将揭示 增强子处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 相对于底层调控DNA序列定位。综合起来，这些目标将 阐明了TF如何开拓染色质景观增强子激活，从而加深了该领域的 了解基因调控机制以及调控不当如何导致人类疾病。