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pancreatic differentiation 2 protein; protein protein interaction; reconstitution; recruit; tool; transcription factor; unpublished works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.

项目概要 已提出由蛋白质本质无序区域 (IDR) 介导的弱多价相互作用 在空间上将转录机器组织成多组分簇，但我们对如何将其组织成多组分簇知之甚少 这些 IDR 与特定合作伙伴互动以实现功能性成果。由于这些相互作用非常 动态且依赖于簇的它们已被识别蛋白质-蛋白质的传统策略所忽视 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 弱多价相互作用能够产生高度特异性的异型相互作用，从而导致功能性 细胞核的组织。我们的长期目标是了解弱多价相互作用如何组织 转录机制的特定组件，以实现基因激活。此举的目的 赠款的目的是研究MED1的IDR簇介导的相互作用的机制和功能， 介导共激活复合物的最大亚基。 Mediator 是一种桥接 DNA 结合的兆道尔顿复合物 转录因子与激活过程的下游步骤要求它动态地参与 监管机制的许多组成部分。虽然最近的结构研究揭示了 在这个复合体中，负责多价相互作用的域是动态 IDR，并且仍未得到解决。在 特别是 MED1 亚基包含 >600 个氨基酸 c 端 IDR (MED1-IDR) 研究涉及簇的形成。我们的初步数据表明 MED1-IDR 簇选择性分区 转录的正调节因子并排除负调节因子或功能不相关但丰富的核 蛋白质。这些数据使我们推测 IDR 介导的选择性区室化是一种机制 来调节转录。为了检验这个假设，我们将确认并验证这些簇的特异性 - 介导的相互作用（目标 1），表征这些相互作用的特异性背后的分子特征 （目标 2），并研究这些相互作用在各种基因激活模型中的功能（目标 3）。之上 完成这些拟议的研究后，我们将了解弱多价相互作用介导的作用 MED1-IDR 参与转录机器特定组件的组织。这个贡献意义重大 因为它将导致人们对基因激活中普遍但经常被忽视的 IDR 的功能有新的认识。 虽然我们在这里关注 MED1-IDR，但这里开发的工具和方法以及学到的原理可以 应用于基因调控或不断增长的生化列表中涉及的其他弱多价相互作用 由调节器的动态集群调节的过程。