The structural basis of direct glucocorticoid-mediated transrepression

Abstract

A newly discovered negative glucocorticoid response element (nGRE) mediates DNA-dependent transrepression by the glucocorticoid receptor (GR) across the genome and plays a major role in immunosuppressive therapy. The nGRE differs dramatically from activating response elements and the mechanism driving GR binding and transrepression is unknown. To unravel the mechanism of nGRE-mediated transrepression by the glucocorticoid receptor, we characterize the interaction between GR and a nGRE in the thymic stromal lymphopoetin (TSLP) promoter. We show using structural and mechanistic approaches that nGRE binding represents a new mode of sequence recognition by human GR and that nGREs prevent receptor dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the presence of monomeric GR at repressive elements.

Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

“…1A). Residues comprising the GR DBD's dimerization loop, such as Ala458, Gly459, and Arg460, showed significant chemical shift perturbations when bound to (+)GRE DNA but not nGRE DNA, supporting the notion that the GR DBD binds to the TSLP nGRE as two monomers (14). Additionally, NMR peaks for arginine side chains appeared when the DBD was bound to nGRE, indicating altered rates of exchange of DBD-(+)GRE and DBD-nGRE complexes.…”

“…S1) indicate that DBD conformation differs when bound to distinct response elements, and such allosteric changes have been shown to affect transcriptional output (21,25). Molecular dynamics (MD) trajectories followed by community and suboptimal path analysis of the SR-DNA complexes show that AncSR2's daughter proteins in the GR and MR lineage contain more complex community organizations, resulting in diverging allosteric communication upon DNA binding among AncSR2-derived paralogs: at nGREs, the community connecting DBD monomers is much larger in hGR versus AncSR2, indicating that DNA-mediated intermonomer communication and resulting negative cooperativity at nGREs (14) is enhanced in hGR compared with AncSR2 (Fig. 3 C-F).…”

“…This result indicates an additional role for epistatic mutations during steroid receptor DBD evolution: either mutations occurred in the lineage leading to AncGR-but not that to MR-which were necessary for Gly425Ser to yield repression at nGREs, or restrictive mutations occurred in the lineage leading to MR, which prevented Gly425Ser from having that same effect. In either case, amino acid substitutions that do not (14). At nGREs, each DBD monomer communicates through a central community (community C).…”

“…All extant and ancestral proteins (46) is shown (Left); the GR DBD can dimerize on an inverted repeat (+)GRE element (21) to activate transcription (Center) or can bind as two monomers on an nGRE (Right) to repress transcription (14). (B) NMR spectra of 15 N-labeled GR DBD in the absence of DNA (Left) or bound to (+)GRE DNA (Center) or nGRE DNA (Right).…”

“…The structural basis of GR-mediated transcriptional repression has remained less clear; however, recent work has shown that negative glucocorticoid response elements, or nGREs, play a role in GR-mediated transcriptional repression (12). At nGREs, monomeric GR DBD binds to an everted repeat with negative cooperativity (14) (Fig. 1A).…”