Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. 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.

The glucocorticoid and progesterone receptors (GR and PR) are closely related members of the steroid receptor family. Despite sharing similar structural and functional characteristics; the cognate hormones display very distinct physiological responses. In mammary epithelial cells, PR activation is associated with the incidence and progression of breast cancer, whereas the GR is related to growth suppression and differentiation. Despite their pharmacological relevance, only a few studies have compared GR and PR activities in the same system. Using a PR+/GR+ breast cancer cell line, here we report that either glucocorticoid-free or dexamethasone (DEX)-activated GR inhibits progestin-dependent gene expression associated to epithelial-mesenchymal-transition and cell proliferation. When both receptors are activated with their cognate hormones, PR and GR can form part of the same complex according to co-immunoprecipitation, quantitative microscopy and sequential ChIP experiments. Moreover, genome-wide studies in cells treated with either DEX or R5020, revealed the presence of several regions co-bound by both receptors. Surprisingly, GR also binds novel genomic sites in cells treated with R5020 alone. This progestin-induced GR binding was enriched in REL DNA motifs and located close to genes coding for chromatin remodelers. Understanding GR behavior in the context of progestin-dependent breast cancer could provide new targets for tumor therapy.

The mineralocorticoid and glucocorticoid receptors (MR and GR) are evolutionary related nuclear receptors with highly conserved DNA- and ligand-binding domains (DBD and LBD), which determine promiscuous activation by corticosteroid hormones (aldosterone and glucocorticoids) and binding to a shared DNA consensus sequence, the hormone response element (HRE). In addition, MR and GR functionally interact, likely through direct formation of heteromeric complexes, potentially contributing to cell-specific corticosteroid signaling. It has recently been proposed that agonist and DNA binding promote GR self-association in tetramers. Here we investigated MR quaternary arrangement after receptor activation. To that end we used a fluorescence imaging technique, Number & Brightness (N&B) analysis, in a cell system where receptor-DNA interaction can be studied in live cells in real time. Our results show that agonist-bound MR is a tetramer in the nucleoplasm, forming higher order oligomers upon binding to HREs. Antagonists form intermediate quaternary arrangements, suggesting that the formation of large oligomeric complexes is essential for function. We also show that divergence between MR and GR quaternary arrangements are driven by different functionality of multimerization interfaces in the DBD and LBD and their interplay with the N-terminal domain. In spite of contrasting quaternary structures, MR and GR are able to form heteromers. Given the importance of both receptors as pharmacological targets and the differential oligomerization induced by antagonists, our findings suggest that influencing quaternary structure may be important to provide selective modulation of corticosteroid signaling.

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other's dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level.

The prevailing model of steroid hormone nuclear receptor function assumes ligand-induced homodimer formation followed by binding to DNA hormone response elements (HREs). This model has been challenged by evidence showing that the glucocorticoid receptor (GR) forms tetramers upon ligand and DNA binding, which then drive receptor-mediated gene transactivation and transrepression. GR and the closely-related mineralocorticoid receptors (MR) interact to transduce corticosteroid hormone signaling, but whether they share the same quaternary arrangement is unknown. Here, we used a fluorescence imaging technique, Number & Brightness, to study oligomerization in a cell system allowing real-time analysis of receptor-DNA interactions. Agonist-bound MR forms tetramers in the nucleoplasm and higher order oligomers upon binding to HREs. Antagonists form intermediate-size quaternary arrangements, suggesting that large oligomers are essential for function. Divergence between MR and GR quaternary structure is driven by different functionality of known and new multimerization interfaces, which does not preclude formation of heteromers. Thus, influencing oligomerization may be important to selectively modulate corticosteroid signaling.