Comprehensive analysis of epigenetic regulators in their native chromatin context

表观遗传调节因子在其天然染色质环境中的综合分析

• 批准号: 8321675
• 负责人: Mitzi I Kuroda
• 金额: $ 33.74万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-13 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321675
• 关键词: ASCL1 gene; Address; Affinity; Affinity Chromatography; Animal Model; Binding; Biochemical Genetics; Biological Models; Cell model; Chromatin; Complement; Complex; Crude Extracts; DNA; DNA Sequence Analysis; DNA-Directed RNA Polymerase; Development; Disease; Dosage Compensation (Genetics); Drosophila genus; Embryo; Enhancers; Epigenetic Process; Event; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genome; Goals; Histones; Human; Human Development; Lead; Link; Literature; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Modification; Molecular; Nature; Organism; PRC1 Protein; Peptides; Physiology; Polycomb; Polymerase; Proteins; RNA; RNA Interference; RNA analysis; Regulatory Element; Regulatory Pathway; Resolution; Role; Site-Directed Mutagenesis; Transcription Initiation; Untranslated RNA; Whole Organism; Work; crosslink; genetic regulatory protein; genome-wide analysis; heterochromatin-specific nonhistone chromosomal protein HP-1; histone modification; human disease; interest; novel; novel strategies; success

DESCRIPTION (provided by applicant): The genomes of higher organisms are highly annotated by specific chromosomal proteins and histone modifications along active genes, regulatory elements, or silent regions. An ongoing challenge is to decipher the rules that establish and maintain chromatin organization. Classic epigenetic regulators such as the Polycomb group (PcG), the Trithorax group (TrxG), and Heterochromatin Protein 1 (HP1), have profound roles in developmental control of the genome in all higher organisms examined. However, one obstacle to understanding their function has been the trade-off between removing them from the DNA, to allow purification, and the resultant loss of weak or transient interactions with key partners. Our new approach allows us to affinity-purify fragmented chromatin with proteins and RNAs attached, using cross-linking to avoid disruption of weak interactions. Using our approach, the linked DNA, protein, histone peptides, and RNA fractions can be separately analyzed using comprehensive sequencing and mass spectrometry. In Aim 1, a dual tag allows affinity purification from low amounts of relatively crude extracts, giving us the ability to look at early events in the establishment of these key chromatin-associated complexes. In Aim 2, we probe their molecular mechanisms by measuring their impact on RNA polymerase distribution across the genome at high resolution. Success in our studies will be relevant to understanding the establishment of conserved genome organization and function, which is central to normal human development and physiology. PUBLIC HEALTH RELEVANCE: Cellular genomes are organized into active and silent chromatin, characterized by specific DNA, RNA, and protein composition. Perturbation of this organization can lead to aberrant development and diseases such as cancer in humans. The goal of our studies is to understand the rules for establishment and maintenance of chromatin organization, and to document its precise functions in gene regulation.

描述（由申请人提供）： 高等生物的基因组由特定染色体蛋白和沿着活性基因、调控元件或沉默区域的组蛋白修饰高度注释。一个持续的挑战是破译建立和维持染色质组织的规则。经典的表观遗传调节因子，如 Polycomb 组 (PcG)、Trithorax 组 (TrxG) 和异染色质蛋白 1 (HP1)，在所有高等生物体的基因组发育控制中具有深远的作用。然而，理解它们功能的一个障碍是在将它们从 DNA 中去除以进行纯化与由此导致的与关键伙伴的微弱或短暂相互作用的丧失之间进行权衡。我们的新方法使我们能够亲和纯化附着有蛋白质和 RNA 的染色质片段，使用交联以避免弱相互作用的破坏。使用我们的方法，可以使用综合测序和质谱法分别分析连接的 DNA、蛋白质、组蛋白肽和 RNA 片段。在目标 1 中，双标签允许从少量相对粗的提取物中进行亲和纯化，使我们能够观察这些关键染色质相关复合物建立过程中的早期事件。在目标 2 中，我们通过高分辨率测量它们对整个基因组中 RNA 聚合酶分布的影响来探究它们的分子机制。我们研究的成功将有助于理解保守基因组组织和功能的建立，这对于正常人类发育和生理学至关重要。 公共卫生相关性： 细胞基因组分为活性染色质和沉默染色质，其特征在于特定的 DNA、RNA 和蛋白质组成。该组织的扰动可能导致人类发育异常和疾病，例如癌症。我们研究的目标是了解染色质组织的建立和维持的规则，并记录其在基因调控中的精确功能。