Mechanisms of enhancer dynamics and assembly in gene regulation

SUMMARY/ABSTRACT Mechanisms of enhancer dynamics and assembly in gene regulation Effective gene regulation is essential for the proper functioning of cells. Dynamic control of enhancer repertoires drives stage-specific transcription during tissue development and disease progression. The overall objective of our research program is to gain a fundamental understanding of the regulations of enhancer dynamics in response to signaling and their effects on gene regulation, leading to innovative approaches to the prevention and treatment of enhancer-related diseases. We propose the following two themes to pursue this objective from multiple levels: from the enhancer chromatin organization, to the coordination of enhanceosome components, and the molecular interactions that drive enhancer assembly. Supported by the NIGMS R01 grant (R01GM137009) and other grants, we have made significant progress in both themes, and the results gained from our previous studies form the strong foundation of this MIRA research program. Theme 1: To profile the context-specific enhancer dynamics and understand its functional significance. Our previous studies have revealed that enhancer dynamics can be induced by 1) acute hormone stimulations, which can build up the active enhancer machinery in just minutes at many chromatin sites to turn on gene expression, and 2) chronic disease progression towards tamoxifen resistance, which reprograms the ERα cistrome to evade endocrine therapies in breast cancer. With these two established model systems (acute and chronic signaling), we will 1) capture the dynamic enhancer chromatin organization with multi-omics analyses and identify candidate key transcription factors associated with enhancer reprogramming using bioinformatic approaches; 2) define stage-specific enhanceosome components using in vivo proximity-dependent biotin labeling BioID/TurboID technologies; and 3) introduce a series of perturbations to understand how disruption of enhancer dynamics affects transcriptional activation and cell state. Theme 2: To decipher inter- and intra-molecular interactions of enhancer components and their contributions to enhancer assembly. Enhancer activation relies on proper enhancer assembly, but the molecular mechanisms of enhancer assembly (how protein-DNA and protein-protein interactions occur) are unclear. We have revealed important principles of enhancer assembly, including combinatorial interactions of multiple transcription factors on hormone-regulated enhancers, and phase-separated condensation mediated by hormone receptor multivalent interactions. Further studies will employ a diverse set of genetic and genomic approaches, as well as our recently established imaging-based approaches, including the LacO arrays/LacI- fluorescence proteins system and a single-molecular tracking (SMT) imaging system. Specifically, we plan to 1) investigate the behaviors of homotypic and heterotypic multivalent interactions of enhanceosome components and their contributions to enhancer assembly; and 2) dissect the intra-molecular interactions of hormone receptors in response to ligand binding and their roles in regulating enhancer assembly and activity.