Interrogating the Structural Basis of Nuclear Receptor Activation on Chromatin

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PROJECT SUMMARY Nuclear receptors (NRs) are ligand-gated transcription factors that control important biological processes including reproduction, development, metabolism, and immunity. NRs are the target of ~16% of drugs used in the clinic for applications that include birth control, inflammatory disease, and metabolic disorders. NRs are thought to function by altering the dynamics of chromatin to alter expression levels of genes through the recruitment of coregulators, chromatin remodelers, and ultimately through the activation of transcriptional machinery. However, the dynamic nature of NRs and NR complexes has made it challenging to study the molecular mechanisms by which full length proteins and protein assemblies regulate gene expression using traditional structural biology techniques. Despite the central role that NRs play in gene expression activation, our mechanistic understanding of how they regulate target gene programs at the molecular level of chromatin is largely unknown. This is attributed, in part, to the dearth of structural biology data for full-length receptors and the near complete absence of biochemical, biophysical, or structural data on NRs in their functionally relevant chromatin context. Building on established foundations in sponsor’s home laboratory, combined with the applicant’s prior expertise in biochemical and biophysical methods and an intimate understanding of the NR field, this project seeks to determine how a model NR named RORγT assembles with coregulatory proteins SRC3 and p300 to alter chromatin dynamics and post-translational modification status of chromatin, a hallmark of NR target gene activation. The proposed research program has two major goals: (1) to determine how RORγT and coregulatory protein complexes engage with nucleosomes, and (2) to determine how RORγT and coregulator protein complexes modify histones and alter nucleosome dynamics. This will be accomplished using a combination of traditional biochemistry, and state-of-the-art technologies including cryogenic electron microscopy (cryo-EM) and structural proteomics. By studying how these protein complexes assemble and act on nucleosomes, we will gain new insight into how RORγT, and more broadly the NR superfamilty, modulates nucleosome dynamics, modification status, and, ultimately, function in gene regulation. The implications of this work will help us understand how an important drug target functions and will pave the way for future research investigating an important class of drug targets.