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      Live Cell Imaging Unveils Multiple Domain Requirements for In Vivo Dimerization of the Glucocorticoid Receptor

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          Abstract

          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.

          Abstract

          Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

          Author Summary

          The powerful anti-inflammatory and immunosuppressive action of glucocorticoids have made them one of the most prescribed drugs worldwide. Unfortunately, acute or chronic treatment may have severe side-effects. Glucocorticoids bind to the glucocorticoid receptor (GR), a ligand-dependent transcription factor. GR regulates gene expression directly by binding to DNA or indirectly by modulating the activity of other transcription factors. It is currently accepted that the direct pathway is mostly responsible for glucocorticoids side-effects and that the oligomerization state of the GR (whether it is a dimer or a monomer) determines which pathway (direct or indirect) will prevail. Hence, scientists have tried to develop “dissociated ligands” able to specifically activate the GR indirect pathway. In the present work, we employed a novel microscopy method named the number and brightness assay, which measures GR oligomerization state inside the living cell. Our results suggest that—contrary to the established view—there is no clear correlation between the oligomerization state of GR and the mechanistic pathway the receptor will follow upon ligand binding. This discovery presents supporting evidence towards the increasing view of the inherent complexity of glucocorticoid action and might impact future approaches towards the design of safer synthetic glucocorticoids.

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          Most cited references45

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          Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells

          Imaging single fluorescent proteins in living mammalian cells is challenging due to out-of-focus fluorescence excitation by common microscopy schemes. We report the development of a novel fluorescence microscopy method, reflected light sheet microscopy (RLSM), which allows selective plane illumination throughout the nucleus of living mammalian cells, for reducing out-of-focus fluorescence signal. Generation of a thin light sheet parallel to the imaging plane and close to the sample surface is achieved by reflecting an elliptical laser beam incident from the top by 45° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrate the sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determine the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor (ER), enabling us to resolve different modes of DNA binding of GR. Finally, we demonstrate two-color single molecule imaging by observing the spatio-temporal co-localization of two different protein pairs. The combination of our single molecule measurements and statistical analysis reveals dynamic properties of transcription factors in live mammalian cells.
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            Transcription dynamics.

            All aspects of transcription and its regulation involve dynamic events. The basal transcription machinery and regulatory components are dynamically recruited to their target genes, and dynamic interactions of transcription factors with chromatin--and with each other--play a key role in RNA polymerase assembly, initiation, and elongation. These short-term binding dynamics of transcription factors are superimposed by long-term cyclical behavior of chromatin opening and transcription factor-binding events. Its dynamic nature is not only a fundamental property of the transcription machinery, but it is emerging as an important modulator of physiological processes, particularly in differentiation and development.
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              Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA.

              Two crystal structures of the glucocorticoid receptor DNA-binding domain complexed with DNA are reported. The domain has a globular fold which contains two Zn-nucleated substructures of distinct conformation and function. When it binds DNA, the domain dimerizes, placing the subunits in adjacent major grooves. In one complex, the DNA has the symmetrical consensus target sequence; in the second, the central spacing between the target's half-sites is larger by one base pair. This results in one subunit interacting specifically with the consensus target half-site and the other nonspecifically with a noncognate element. The DNA-induced dimer fixes the separation of the subunits' recognition surfaces so that the spacing between the half-sites becomes a critical feature of the target sequence's identity.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                1544-9173
                1545-7885
                March 2014
                18 March 2014
                : 12
                : 3
                : e1001813
                Affiliations
                [1 ]Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, NIH, Bethesda, Maryland, United States of America
                [2 ]Department of Biological Chemistry, School of Sciences (FCEN), University of Buenos Aires (UBA), Buenos Aires, Argentina
                [3 ]IFIBYNE-CONICET, School of Sciences (FCEN), University of Buenos Aires (UBA), Buenos Aires, Argentina
                [4 ]Department of Organic Chemistry/UMYMFOR-CONICET, School of Sciences (FCEN), University of Buenos Aires (UBA), Buenos Aires, Argentina
                [5 ]Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, United Kingdom
                [6 ]Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
                [7 ]Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, United States of America
                University of California, San Francisco, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: DMP LDA VL GLH AP. Performed the experiments: DMP MFO MS JRP. Analyzed the data: DMP MFO MS JRP LG VL. Contributed reagents/materials/analysis tools: RLS TAJ PRM JDA SG GB VL GLH AP. Wrote the paper: DMP LDA GLH AP.

                Article
                PBIOLOGY-D-13-04323
                10.1371/journal.pbio.1001813
                3958349
                24642507
                329f7918-d8cd-4e0a-bb69-67d77727b840
                Copyright @ 2014

                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 5 November 2013
                : 6 February 2014
                Page count
                Pages: 14
                Funding
                This research was supported by grants from CONICET (PIP 112-200801-00859), Agencia Nacional de Promociones Científicas y Técnicas (BID.PICT 2011-1321), University of Buenos Aires (UBACyT), and the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                DNA
                Nucleic Acids
                Cell Biology
                Signal Transduction
                Cell Signaling
                Nuclear Receptor Signaling
                Molecular Cell Biology

                Life sciences
                Life sciences

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