Role of nucleosome architecture in cellular reprogramming

核小体结构在细胞重编程中的作用

• 批准号: 10567857
• 负责人: Motoki Takaku
• 金额: $ 29.61万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10567857
• 关键词: 3-Dimensional; Address; Affect; Algorithms; Architecture; Binding; Binding Sites; Biological; Biological Models; Breast Cancer Cell; CHD4 gene; CRISPR/Cas technology; Cells; Cellular biology; Chromatin; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cryoelectron Microscopy; DNA; Data; Enabling Factors; Enhancers; Enzymes; Epigenetic Process; Epithelium; GATA3 gene; Gene Activation; Genes; Genome; Genomic DNA; Goals; Grant; Higher Order Chromatin Structure; Histones; Human; Human Genome; Knock-out; Location; MDA MB 231; Malignant Neoplasms; Maps; Mediating; Mesenchymal; Methods; Modeling; Molecular; Molecular Conformation; Mutation; Nucleosomes; Positioning Attribute; Process; Proteins; Resolution; Role; SMARCA4 gene; Site; Structure; System; Techniques; Therapeutic; Thinness; Transcript; breast tumorigenesis; cellular development; chromatin remodeling; cofactor; epithelial to mesenchymal transition; genomic data; genomic locus; human disease; induced pluripotent stem cell; insight; malignant breast neoplasm; novel; novel strategies; predictive modeling; prevent; programs; transcription factor; triple-negative invasive breast carcinoma

PROJECT SUMMARY Enhancer formation at precise chromatin loci by transcription factors enables appropriate gene activation to biological demand. This essential process is challenged by the millions of matches to transcription factor binding motifs present in the human genome. Chromatin structure is also known to act as a physical barrier for many transcription factors. How do transcription factors overcome chromatin barriers and selectively activate appropriate genomic loci while they are competing against chromatin barriers? The answer to this fundamental question is still largely unknown. Recently, a subset of transcription factors, so-called pioneer factors, have been identified as essential proteins required to modify chromatin accessibility during cellular reprogramming. Unlike other transcription factors, pioneer factors are capable of binding to nucleosomes at closed (inactive) chromatin sites and inducing chromatin opening. Since nucleosome formation was thought to act as a physical barrier, identification of pioneer factors’ action on chromatin is fundamental to understanding the cellular reprogramming processes. Misregulation of pioneer factors is associated with various human diseases including cancer. Therefore, there is a critical need to identify the molecular mechanisms underlying pioneer factor-induced cellular reprogramming. Our long-term goal is to elucidate the molecular mechanisms of transcription factor-induced cellular reprogramming. We previously demonstrated that GATA3 acts as a pioneer factor that can directly bind closed chromatin and activate epithelial marker genes during the mesenchymal-to-epithelial transition (MET) in triple-negative breast cancer cells. Using this cellular reprogramming model system, we further identified the unique nucleosome positioning enriched at GATA3 target loci. Our Cryo-EM structure analysis of the GATA3-nucleosome complex revealed that the motif recognition by GATA3 on the nucleosome differs from histone-free DNAs. Chromatin structural analysis during MET also identified that chromatin opening and de novo enhancer formation by GATA3 is site-specific and only observed at a subset of GATA3 binding sites. Based on this evidence, we hypothesize that nucleosome positioning and structure at the pioneer factor GATA3 binding site dictate the readout (chromatin opening and gene activation) of GATA3 binding and is directly involved in MET. The primary goals of this application are to: (1) identify specific nucleosome positioning and conformation that are essential for successful GATA3- induced cellular reprogramming; (2) identify the roles of chromatin remodeling enzymes during GATA3- induced MET, and; (3) discover minimum chromatin components for GATA3-induced enhancer formation. We will use several novel approaches including high-resolution mapping methods for nucleosome positioning and transcription factors’ footprint. These results will provide a basis for understanding cellular reprogramming processes induced by transcription factors.

项目摘要 通过转录因子以精度染色质基因座形成增强剂，使适当的基因激活 生物需求。这一基本过程受到数百万次转录的挑战 人类基因组中存在的因子结合基序。染色质结构也被称为 许多转录因子的物理障碍。转录因子如何克服染色质屏障 并在与染色质屏障竞争时有选择地激活合适的基因组局部？ 这个基本问题的答案在很大程度上是未知的。最近，转录子集 因素，所谓的先驱因素，已被确定为修饰染色质所需的必需蛋白质 细胞重编程期间的可访问性。与其他转录因素不同，先锋因素能够 在封闭（非活性）染色质位点和诱导染色质开口处与核小体结合。自从 核小体形成被认为是物理障碍，对先锋因素对行动的识别 染色质是理解细胞重编程过程的基础。误导 先锋因素与包括癌症在内的各种人类疾病有关。因此，有一个关键 需要确定先锋因子诱导的细胞重编程的分子机制。 我们的长期目标是阐明转录因子诱导的细胞的分子机制 重新编程。我们先前证明了GATA3是可以直接结合的先锋因素 封闭的染色质并激活间质至上皮过渡期间上皮标记基因 （Met）在三阴性乳腺癌细胞中。使用此蜂窝重编程模型系统，我们进一步 鉴定出富含GATA3目标基因座的独特核小体定位。我们的冷冻EM结构 对GATA3-核体复合体的分析表明，GATA3在 与无组蛋白DNA的核小体差异。 MET期间的染色质结构分析也确定 通过GATA3的染色质开放和从头增强子形成是特定于位点的，仅在A GATA3结合位点的子集。基于此证据，我们假设核小体定位和 先锋因子GATA3结合位点的结构决定了读数（染色质开口和基因 GATA3结合的激活），直接参与MET。该应用程序的主要目标是： （1）确定对成功GATA3-至关重要的特定核病组定位和考虑因素 诱导细胞重编程； （2）确定染色质重塑酶在GATA3-中的作用 诱发的人； （3）发现GATA3诱导的增强剂的最小染色质成分 形成。我们将使用几种新颖的方法，包括高分辨率映射方法 核小体定位和转录因子的足迹。这些结果将为 了解转录因子诱导的细胞重编程过程。