Project Summary (Parent Grant R01GM147583) 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.