Nuclear Organization and Function

核组织和功能

• 批准号: 10083368
• 负责人: Victor G. Corces
• 金额: $ 43.11万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10083368
• 关键词: 3-Dimensional; Affect; Biological Models; Cell Differentiation process; Cell Nucleus; Cells; Cellular biology; Chromatin; Chromatin Fiber; Complex; Computational Biology; Data; Dimensions; Disease; Drosophila genus; Enhancers; Environment; Epigenetic Process; Eukaryota; Frequencies; Gene Expression; Gene Expression Regulation; Genetic; Genetic Diseases; Genetic Materials; Genetic Transcription; Genome; Hi-C; Homeostasis; Human Genetics; Knowledge; Logic; Machine Learning; Malignant Neoplasms; Mammals; Nuclear; Pancreas; Proteins; Resolution; Role; Site; Somatic Cell; Testing; Three-dimensional analysis; Tissues; Transcription Process; Transcriptional Regulation; Vertebrates; Work; base; cohesin; computerized tools; epigenomics; experimental study; human disease; human embryonic stem cell; interdisciplinary approach; mammalian genome; novel; promoter; protein complex; scaffold; stem cell differentiation; stem cell therapy

PROJECT SUMMARY. Proper regulation of gene expression is essential for cell differentiation and homeostasis. Most of our understanding of the mechanisms that control the transcription process comes from studies of the one-dimensional genome i.e. the 10 nm chromatin fiber. However, the genome is folded in the three-dimensional (3D) nuclear space, and the relationship between this organization and gene expression is poorly understood. Using Drosophila as a model system, where it is feasible to obtain 250 bp resolution Hi-C data, we have found that the genome is folded into only one type of domain, which we call compartmental domains. These domains precisely correlate with the transcriptional state of their sequences. Compartmental domains are also found in other lower eukaryotes. Based on this, we propose that compartmental domains represent an evolutionarily conserved principle of genome 3D organization. Drosophila and lower eukaryotes either lack CTCF or this protein is unable to stop cohesin extrusion. However, CTCF can interfere with the progression of cohesin extrusion in vertebrates, which in turn affects other types of interactions in the genome. Here we suggest extending concepts learned from the analysis of 3D organization in Drosophila to mammals by proposing an ambitious and substantive multi-disciplinary approach combining genetics, epigenomics, computational biology, and differentiation of human embryonic stem cells (hESC) into disease-relevant tissues. The hypothesis underlying the proposed experiments is based on the idea that, rather than the prevalent view of large compartments containing smaller TADs, the mammalian genome is organized by conserved principles into relatively small compartmental domains. Cohesin extrusion operates on top of the compartmental domain scaffold and affects its organization. To test this novel hypothesis, we will deplete specific proteins present in complexes required for various aspects of the transcription process. We will also deplete protein complexes responsible for H3K27me3- and H3K9me3-dependent silencing. We will then use Micro-C XL to obtain very high-resolution interaction data and examine effects of protein depletion on the formation of self-interacting domains and in the interactions between these domains. These effects will be examined in the presence and absence of cohesin in order to understand the contribution of loop extrusion to enhancer-promoter interaction frequency. We will examine the predictability of 3D genome organization from one-dimensional epigenetic information using machine learning computational tools. We will study the logic of CTCF loop formation by analyzing the local chromatin environment around CTCF sites able or unable to form loops of different strengths using a new computational tool we have developed. Principles learned from these experiments will be tested by analyzing changes in 3D organization and their relationship to gene expression during the differentiation of hESCs into pancreatic cells. Results from this work will fill critical gaps in our understanding of the relationship between 3D chromatin organization and transcription, and its possible role in human disease.

项目摘要。基因表达的正确调控对于细胞分化和 体内平衡。我们对控制转录过程的机制的大部分理解来自 一维基因组（即 10 nm 染色质纤维）的研究。然而，基因组折叠在 三维（3D）核空间，该组织与基因表达之间的关系为 不太了解。使用果蝇作为模型系统，可以获得 250 bp 分辨率的 Hi-C 数据中，我们发现基因组仅折叠成一种类型的结构域，我们称之为区室 域。这些结构域与其序列的转录状态精确相关。隔室式 结构域也存在于其他低等真核生物中。在此基础上，我们提出分区域 代表基因组 3D 组织的进化保守原则。果蝇和低等真核生物 要么缺乏 CTCF，要么该蛋白无法阻止粘连蛋白挤出。然而，CTCF 可能会干扰 脊椎动物中粘连蛋白挤出的进展，进而影响基因组中其他类型的相互作用。 在这里，我们建议将从果蝇 3D 组织分析中学到的概念扩展到哺乳动物 通过提出一种雄心勃勃且实质性的多学科方法，结合遗传学、表观基因组学、 计算生物学，以及人类胚胎干细胞（hESC）分化为疾病相关组织。 所提出的实验的假设是基于这样的想法，而不是普遍的观点 哺乳动物基因组由包含较小 TAD 的大区室组成，按照保守原则进行组织 分成相对较小的分区域。粘连蛋白挤出在区室域顶部进行 支架并影响其组织。为了测试这个新假设，我们将耗尽存在于 转录过程各个方面所需的复合物。我们还将耗尽蛋白质复合物 负责 H3K27me3 和 H3K9me3 依赖性沉默。然后我们将使用 Micro-C XL 来获得非常 高分辨率相互作用数据并检查蛋白质消耗对自相互作用形成的影响 领域以及这些领域之间的相互作用。这些影响将在存在和 缺乏粘连蛋白，以了解环挤出对增强子-启动子相互作用的贡献 频率。我们将从一维表观遗传研究 3D 基因组组织的可预测性 使用机器学习计算工具的信息。我们将通过以下方式研究 CTCF 循环形成的逻辑 分析 CTCF 位点周围能够或不能形成不同环的局部染色质环境 使用我们开发的新计算工具的优势。从这些实验中学到的原理将 通过分析 3D 组织的变化及其与基因表达的关系来进行测试 hESC 分化为胰腺细胞。这项工作的结果将填补我们理解的关键空白 3D 染色质组织与转录之间的关系及其在人类疾病中的可能作用。