Organization of transcriptional machinery by weak multivalent interactions

通过弱多价相互作用组织转录机制

• 批准号: 10684132
• 负责人: Benjamin Sabari
• 金额: $ 33.85万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684132
• 关键词: Amino Acid Substitution; Amino Acids; Architecture; Back; Binding; Biochemical Process; Biological Assay; Biological Process; Biotinylation; C-terminal; Cell Nucleus; Cells; Complement; Complex; DNA Binding; DNA Polymerase II; Data; Defect; Development; Developmental Process; Disease; Doctor of Philosophy; Exclusion; Fluorescence; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Grant; Investigation; Knock-out; Knowledge; Learning; Libraries; Link; Mass Spectrum Analysis; Mediating; Mediator; Methods; Microscopy; Modeling; Molecular; Nature; Nuclear Extract; Nuclear Proteins; Process; Protein Fragment; Protein Region; Proteins; Proteomics; Publishing; RNA; Reporter; Role; Specificity; Techniques; Testing; Time; Transcription Process; Transcriptional Activation; Work; crosslink; epigenomics; experimental study; functional outcomes; genomic locus; human disease; insight; lipid biosynthesis; negative elongation factor; novel; pancreatic differentiation 2 protein; protein protein interaction; reconstitution; recruit; tool; transcription factor; unpublished works

Project Summary Weak multivalent interactions mediated by intrinsically disordered regions (IDRs) of proteins have been proposed to spatially organize the transcriptional machinery into multi-component clusters, yet we know little about how these IDRs interact with specific partners to enable functional outcomes. As these interactions are highly dynamic and cluster-dependent they have been overlooked by conventional strategies to identify protein-protein interactions. Our preliminary data support our overarching hypothesis that in the context of higher-order clusters weak multivalent interactions are capable of highly specific hetero-typic interactions leading to functional organization of the nucleus. Our long-term objective is to understand how weak multivalent interactions organize specific components of the transcriptional machinery in order to enable gene activation. The objective of this grant is to investigate the mechanism and function of cluster-mediated interactions of the IDR of MED1, the largest subunit of the mediator coactivator complex. Mediator is a megadalton complex that bridges DNA-binding transcription factors with downstream steps of the activation process requiring it to engage dynamically with many components of the regulatory machinery. While recent structural studies have revealed the architecture of this complex, the domains responsible for multivalent interactions are dynamic IDRs and remain unresolved. In particular the MED1 subunit contains a >600 amino acid c-terminal IDR (MED1-IDR) which previously published studies implicate in cluster formation. Our preliminary data show that MED1-IDR clusters selectively partition positive regulators of transcription and exclude negative regulators or functionally unrelated yet abundant nuclear proteins. These data lead us to hypothesize that IDR-mediated selective compartmentalization is a mechanism to regulate transcription. To test this hypothesis we will confirm and validate the specificity of these cluster- mediated interactions (Aim 1), characterize the molecular features underlying specificity of these interactions (Aim 2), and investigate the function of these interactions in various models of gene activation (Aim 3). Upon completion of these proposed studies, we will understand the role of weak multivalent interactions mediated by MED1-IDR in organization of specific components of the transcriptional machinery. This contribution is significant as it will lead to a new appreciation for the function of the prevalent yet often overlooked IDRs in gene activation. While we focus here on MED1-IDR, the tools and methods developed and the principles learned here can be applied to other weak multivalent interactions involved in gene regulation or the growing list of biochemical process regulated by dynamic clustering of regulators.

项目摘要 已经提出了由本质上无序区域（IDR）介导的蛋白质的弱多价相互作用 要将转录机械组织到多组分群集中，但我们对如何如何了解 这些IDR与特定合作伙伴互动以实现功能结果。因为这些互动很高 传统策略鉴定蛋白质 - 蛋白质蛋白 互动。我们的初步数据支持我们的总体假设，即在高阶集群的背景下 弱的多价相互作用能够具有高度特异性的异质相互作用，从而导致功能 核组织。我们的长期目标是了解弱的多价互动如何组织 转录机械的特定成分，以实现基因激活。这个目的 授予是为了研究群集介导的Med1 IDR相互作用的机制和功能， 介体共激活因子络合物的最大亚基。介体是一个桥接DNA结合的Megadalton综合体 转录因子具有激活过程的下游步骤，要求其动态互动 监管机械的许多组件。虽然最近的结构研究揭示了 该复合物负责多价交互的域是动态IDR，并且仍未解决。在 特别是Med1亚基包含> 600氨基酸C末端IDR（MED1-IDR） 研究与群集形成有关。我们的初步数据显示Med1-IDR簇有选择地分区 转录的积极调节剂，排除负调节器或功能无关但丰富的核 蛋白质。这些数据使我们假设IDR介导的选择性隔室化是一种机制 调节转录。为了检验这一假设，我们将确认并验证这些群集的特异性 介导的相互作用（AIM 1）表征了这些相互作用的分子特征 （AIM 2），并研究这些相互作用在各种基因激活模型中的功能（AIM 3）。之上 这些提出的研究的完成，我们将了解由弱的多价相互作用的作用 Med1-IDR在转录机械的特定组件组织中。这项贡献很重要 因为这将导致对基因激活中普遍但经常被忽略的IDR的功能的新欣赏。 虽然我们将重点放在Med1-IDR上，但开发了工具和方法，这里学到的原理可以是 应用于基因调节或生化列表中涉及的其他弱的多价相互作用 通过调节器的动态聚类调节过程。