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Organization of transcriptional machinery by weak multivalent interactions

通过弱多价相互作用组织转录机制

基本信息

批准号：
10886179
负责人：
Benjamin Sabari
金额：
$ 7.65万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/10886179
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novel pancreatic differentiation 2 protein protein protein interaction reconstitution recruit tool transcription factor unpublished works

项目摘要

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蛋白质固有无序区(IDR)介导的弱多价相互作用在空间上将转录机制组织成多组分的集群，但我们对如何这些IDR与特定的合作伙伴互动，以实现职能成果。因为这些相互作用是高度传统的鉴定蛋白质-蛋白质的策略忽视了它们的动态和依赖于簇的特性互动。我们的初步数据支持我们的主要假设，即在更高阶星系团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，从而导致功能性原子核的组织。我们的长期目标是了解弱多价相互作用是如何组织的转录机制的特定组成部分，以实现基因激活。这样做的目的是 GRANT是为了研究MED1IDR簇介导的相互作用的机制和功能，介体共激活物复合体的最大亚基。Mediator是一个连接DNA结合的百万级复合体转录因子，激活过程的下游步骤需要它动态地与监管机制的许多组成部分。虽然最近的结构研究揭示了这种复杂的、负责多价相互作用的结构域是动态的IDR，并且仍然未被解析。在……里面特别是，MED1亚单位含有先前发表的600个氨基酸的c-末端IDR(MED1-IDR) 研究表明，这与星系团的形成有关。我们的初步数据显示，MED1-IDR星团选择性地划分正性转录调节因子，排除负性调节因子或功能无关但丰富的核蛋白质。这些数据让我们假设，IDR介导的选择性划分是一种机制来调节转录。为了验证这一假设，我们将确认和验证这些星系团的特异性- 中介相互作用(目标1)，描述这些相互作用特有的分子特征 (目标2)，并研究这些相互作用在各种基因激活模式中的作用(目标3)。vt.在.的基础上完成这些建议的研究，我们将了解弱多价相互作用在转录机器的特定组件的组织中的MED1-IDR。这一贡献是巨大的因为这将导致对普遍但经常被忽视的IDR在基因激活中的作用的新的认识。虽然我们在这里关注的是MED1-IDR，但开发的工具和方法以及在这里学到的原则可以是用于其他涉及基因调控的弱多价相互作用或日益增长的生物化学由调节器的动态聚类来调节的过程。