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 mouse mammary tumor virus acquires a highly reproducible chromatin structure when integrated into cellular DNA. Previous studies have suggested that the LTR is arranged as a series of six phased nucleosomes, that occupy specific positions on the LTR. On the basis of nucleosome reconstitution studies using DNA from the B region of the LTR, it has been argued that this sequence directs a uniquely positioned nucleosome. Here we demonstrate in vitro that reconstituted B region nucleosomes adopt at least five distinct translational positions in two rotational frames on a 206 bp fragment of DNA. We have resolved an initial reconstitute into its component species using nondenaturing gel electrophoresis, and precisely mapped the positions of each species using a hydroxyl radical footprinting assay. To confirm the nucleosome positions determined with the hydroxyl radical assay, nucleosome boundaries were mapped using exonuclease III. Comparison of the results from the hydroxyl radical footprinting and exonuclease III assays revealed a symmetrical pattern of overdigestion by exonuclease III which made unequivocal determination of nucleosome boundaries dubious. We conclude that the general use of exonuclease III to map the positions of nucleosomes may lead to incorrect assignment of position, and that assignment of position through the determination of the nucleosome pseudo-dyad from hydroxyl radical footprinting data represents a superior method of analysis.

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