Interrogating the Role of Chromatin in Nuclear Receptor Structure and Function

About This Grant

PROJECT SUMMARY/ABSTRACT The nuclear receptors (NRs) named retinoic acid receptor-related orphan receptor γ isoform 2 (RORγt) and glucocorticoid receptor (GR) are powerful transcription factors that mediate central functions of the adaptive and innate immune system, respectively. NRs are a major family of drug targets and mechanistic studies are needed to better understand how drugs impact NR function. NRs function by binding to their respective small molecules, localizing to specific DNA sequences in the genome called response elements, and then by recruiting coregulatory protein complexes that (1) modify histones, (2) promote chromatin remodeling, and (3) recruit transcription initiation machinery. This proposal seeks to unveil the mechanisms of RORγt- and GR-mediated histone acetylation – a hallmark of NR target gene activation. This will be done using a combination of chromatin biochemistry, structural biology, and functional genomic analyses. The underlying hypothesis is that the chromatin context provides important cues for how target genes are regulated by RORγt and GR, as well as by their associated coregulatory protein complexes. In support of the hypothesis, exciting preliminary data is disclosed showing how the precise positioning of the DNA response element on the nucleosome (the fundamental unit of chromatin) dictates if and how RORγt and GR engage with chromatin. Going further, the first structure of RORγt bound to the nucleosome is presented, which shows an unexpected and novel positioning of domains on the nucleosome. This project will extend from these studies by building up RORγt:coregulator complexes using model coregulatory proteins named SRC3 and p300. Previous biochemical studies with NRs have reduced these large multidomain proteins to small peptide fragments or have failed to achieve high- resolution. Solving structures of full length SRC3 and p300 with RORγt will define how NRs and chromatin direct these important proteins to their substrates. In addition, this project aims to complement in vitro biochemical and biophysical analyses with novel functional analyses of RORγt in a relevant primary T cell model biological system. Specifically, the candidate (Tim) will be trained to isolate and culture primary naïve T cells as well as to perform a technique called CUT&RUN. The goal of these experiments is to learn how RORγt and coregulators work together to drive the expression of pro-inflammatory IL-17 cytokine in naïve T cells. These results will uncover the specific changes in chromatin PTMs and nucleosome positioning during RORγt-mediated activation of its target genes. Finally, in the independent phase, the project aims to extend the methodologies that Tim has learned during his postdoctoral training to GR and its fundamental role in macrophage biology. This extension would form a new biological system that Tim will investigate in his future career. Overall, this innovative and interdisciplinary research strategy will answer important biological questions regarding NR structure and function, with significant impact on human health. The career development plan will take place at The Salk Institute and provide an important opportunity for training and that will prepare Tim to start independent research career.