Project Summary/Abstract Transcription is a fundamental cellular process whose proper regulation is essential to establishment and maintenance of healthy cell states. As with many regulatory processes in the cell, transcription is now understood to involve the dynamic formation and dissolution of large assemblies of protein and RNA molecules called biomolecular condensates. Our research program is focused on three goals at the intersection of transcription and condensates that we believe will provide important new insights into gene regulation and fill important gaps in our understanding of condensates and their regulation. Goal 1) We will test the hypothesis that many long noncoding RNAs (lncRNAs) regulate transcriptional condensates at nearby genes. Condensates are formed by an ensemble of low-affinity molecular interactions and RNA can be a powerful regulator of condensate dynamics. Thousands of lncRNA species are expressed in any one cell type, but the functions of the vast majority of these RNA molecules are not known. Most lncRNAs are transcribed within 10kb of protein coding genes and appear to accumulate at those loci, suggesting that many of these RNAs function to tune the expression of local protein coding genes by affecting the dynamics of local condensate formation and dissolution. Goal 2) We will test the hypothesis that condensate immiscibility contributes to the functional separation of active and silent chromatin. The nuclear architecture of a cell involves transcriptionally active and inactive compartments, and current evidence indicates that the two compartments form separate condensates. We have observed that condensates formed by regulators of active and silent genes are immiscible and postulate that this property contributes to the functional separation of active and inactive compartments in the nucleus of mammalian cells. Goal 3) We will explore the physicochemical environments of nuclear condensates with the goal of determining the types of chemistries that distinguish diverse condensates. A major issue in condensate biology is the extent to which the chemical environments of diverse condensates enable biological specificity. Our evidence indicates that small molecules can be used to probe the internal chemical environment that governs the behavior of condensates and thus teach us about the internal chemistry of diverse condensates that may enable biological specificity. This information may also provide insights into the chemical features that selectively concentrate small molecules in specific condensates, which may enable future advances in drug design for targets that reside in specific condensates. While conducting these studies, we will continue to identify protein and RNA components of euchromatic and heterochromatin condensates and to invest in assays of condensate dynamics and transcriptional output. We will also continue to train and mentor diverse young scientists in an environment that facilitates collaboration...