Summary The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Furthermore, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligandregulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors. Steroid receptors are transcription factors regulated by physiological stimuli that dynamically bind to chromatin and control complex biological pathways (1). In particular, the glucocorticoid receptor (GR) is essential for life and is one of the most targeted proteins in the pharmacological industry due to its powerful antiinflammatory and immunosuppressive activities (2). Current pharmaceutical approaches are based on a recently challenged (3) binary model wherein direct binding of GR dimers and indirect binding of GR monomers via other proteins determine the transcriptional output (4).Upon hormone activation, GR associates to a subset of glucocorticoid response elements (GREs) across the genome, depending on the accessibility of the chromatin landscape (5). GR is a modular protein organized into three structural and functional domains; the N-terminal ligand-independent activation function-1 domain (NTD), the central DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD) (6). GR, and all steroid receptors, are widely believed to bind DNA directly as homodimers (7). However, this paradigm has been established exclusively from in vitro studies, working mostly with the DBD fragment (8-10), only using the whole GR protein in rare cases (11,12). The small number of experiments performed in live cells only addresses the entire nuclear population, lacking specific information regarding the GR fraction bound to chromatin (13-16). Furthermore, these studies were unable to discriminate between dimers or higher oligomeric states.For the present study, we combine an experimental model where GR-DNA interaction can be observed in real time with techniques that allow the quantification of the oligomeric state of proteins inside living...

The glucocorticoid receptor's oligomerization state is revealed to not correlate with its activity; this challenges the current prevailing view that this state defines its transcriptional activity.