Regulatory Mechanisms Linking Spatial Gene Control and Genome Organization

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PROJECT SUMMARY Cellular homeostasis is maintained by signaling events that turn on downstream transcription factors to rapidly activate or repress hundreds of genes. The application of next-generation sequencing technologies and super- resolution imaging in the past decade has revealed the central role of non-coding regulatory elements called enhancers in the spatio-temporal control of mammalian gene expression. Recent evidence indicates that acute signaling events assemble the enhancer-bound transcriptional complex as membraneless compartments known as condensates. An important question is how these organelles interact with larger nuclear bodies to shape three-dimensional genome organization and spatial gene control. We have identified three critical challenges that limit our understanding of spatial gene control. Our research program is focused on solving these challenges to fill the critical knowledge gaps in signal-induced gene regulation. Challenge 1) Understand how enhancer communication works. In addition to controlling primary target genes, several pathway-specific enhancers engage in long-distance interactions. Our data indicate that enhancer hubs facilitate genome-wide coordination of signaling programs. The prevalence and regulatory features of this mechanism in signal-activated gene programs are just beginning to be explored. We will use prototypic type I and type II nuclear hormone receptors to study the role of enhancer hubs in transcription coordination. Challenge 2) Elucidate the interaction of non- coding genome and disordered proteome in gene control. We have reported that the ligand-induced enhancer condensates are composed of proteins with intrinsically disordered regions (IDR) and RNAs. Although a common feature of many transcriptional complexes, the molecular and enzymatic regulation of the assembly, dissolution, and material properties of transcriptional condensates are largely unknown. We will use single-cell CRISPR screening strategies, mutational scans, biophysical and genomics assays to identify enzymes controlling signaling-activated enhancers by modifying the IDR structure. Challenge 3) Unravel the cell biological basis of the spatial genome organization. The eukaryotic genome is compartmentalized based on the transcriptional states of the chromatin. Our data indicate that several nuclear architectural structures and transcriptional condensates act as solid-state anchors to facilitate long-distance enhancer interaction and organize chromatin architecture. To gain mechanistic insights into the role of common nuclear bodies in genome organization, we will employ genetic strategies to transiently degrade these organelles and assess the impact on three-dimensional chromatin structure and enhancer function using imaging and genomics tools. By addressing these questions at the intersection of non-coding genome, nuclear condensates, and gene regulation, we will unravel a cell biological basis of spatial gene control. This research will also contribute to developing innovative research tools to fill the knowledge gaps in the regulatory roles of the non-coding genome while facilitating the training and mentoring of the next generation of researchers in multidisciplinary science.