Comprehensive analysis of epigenetic regulators in their native chromatin context

描述（由申请人提供）： 高等生物的基因组由特定的染色体蛋白和组蛋白修饰沿着活性基因、调节元件或沉默区域高度注释。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），TrxG组（TrxG）和异染色质蛋白1（HP1），在所有高等生物的基因组发育控制中具有深远的作用。然而，理解它们的功能的一个障碍是将它们从DNA中去除以允许纯化，以及由此导致的与关键伙伴的弱或短暂相互作用的损失之间的权衡。我们的新方法使我们能够亲和纯化附着有蛋白质和RNA的片段化染色质，使用交联来避免弱相互作用的破坏。使用我们的方法，连接的DNA，蛋白质，组蛋白肽和RNA组分可以分别使用综合测序和质谱分析。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的早期事件。在目标2中，我们通过高分辨率测量它们对RNA聚合酶在基因组中分布的影响来探索它们的分子机制。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，这对正常的人类发育和生理至关重要。 公共卫生相关性： 细胞基因组被组织成活性和沉默的染色质，其特征在于特定的DNA、RNA和蛋白质组成。对这个组织的干扰会导致人类的异常发育和疾病，如癌症。我们的研究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。

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.