OR26-6 Glucocorticoid Receptor Quaternary Structure Drives Chromatin Occupancy and Transcriptional Outcome

Abstract

The multimeric structure adopted by transcription factor complexes at their genomic sites of interaction is a longstanding problem in transcription biology. Information bearing on this question is often inferred from indirect data sources. Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Glucocorticoid receptor (GR)’s dimeric/monomeric status is widely believed to drive pharmacological output of synthetic glucocorticoids. A widely discussed model suggests that dimeric GR regulates unfavorable metabolic pathways, while monomeric GR is responsible for anti-inflammatory activities. However, recent studies have indicated that dimeric GR is equally active in anti-inflammatory activities. Furthermore, quantitative fluorescence microscopy techniques in live cells show that the GR likely forms tetramers on enhancers during gene activation. These results suggest that higher oligomerization states are important for the gene regulatory responses of GR. To unveil the relationship between GR’s oligomerization and transcriptional outcome, we developed a GR null cell system to study how wild type GR and three different receptor oligomerization mutants bind chromatin and affect transcription at the genomic level. All mutants display unique quaternary structure characteristics; DNA-binding domain dimerization deficient (GRdim, A465T), monomeric (GRmon, A465T/I634A), and tetrameric (GRtetra, P481R) receptor. GRtetra mimics the DNA-bound conformation and forms tetramers independent of DNA binding. Integration of ChIP-seq, ATAC-seq, and RNA-seq data indicates that GR must form dimers to bind even pre-accessible chromatin, as monomeric GR is essentially a non-functional receptor. In comparison, GRtetra, acts as a super receptor by binding glucocorticoid response elements not accessible to the wild type receptor. This super receptor behavior can be observed in two distinct cell types. The sites occupied only by GRtetra in a given cell line are bound by the wild type GR in numerous other cell types, such as liver, macrophages, and pre-adipose cells. This suggest that the sites are authentic response elements. In addition, GRtetra both induces and, more importantly, represses more genes in response to hormone than the wild type GR. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher order structure that drives both the activating and repressive actions of glucocorticoids. This has important implications for the therapeutic uses of steroid hormones and the goal of finding selective anti-inflammatory drugs that do not create unwanted side-effects.