The Structural Basis of RORγt Binding to Chromatin 9093

Abstract Description The retinoic acid receptor-related orphan receptor γ isoform 2 (RORγt) is a transcription factor that plays critical roles in adaptive immunity, autoimmune disease, and is best known as the so called ‘master regulator’ of IL-17 expressing T helper cells (Th17). RORγt binds to specific DNA sequences in the genome through its DNA binding domain and recruits coregulatory proteins through its ligand binding domain. The objectives of our research program is to determine (1) how RORyt engages chromatin and (2) the functional implications of chromatin engagement. To address this question, we adapted a biochemical assay called selected engagement of nucleosomes coupled with sequencing (SeEN-seq) to profile how RORγt binds to nucleosomes that contain long DNA extensions, as a model of chromatin. The results from SeEN-seq show that RORγt preferentially engages the entry/exit sites of nucleosomes, where DNA is loosely tethered, but not the core DNA around the histone octamer. We then solved a structure of RORγt bound to the entry site of the nucleosome using cryogenic electron microscopy (cryo-EM). The structure delineates the configuration of the DBD when bound to the response element and also reveals an unanticipated arrangement of the LBD over the histone octamer. The structure-based model and follow up biochemical analyses help to make inferences of how the biochemical activities of RORγt help explain observed chromatin binding preferences. Funding Sources F32 GM148049 U01 AI136680 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)