title: Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.
abstract: The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 μM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.
authors: Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary.
authors: Nikoletta
authors: N
authors: Szalóki
authors: German Cancer Research Center (DKFZ), Biophysics of Macromolecules, Heidelberg, Germany.
authors: Jan Wolfgang
authors: JW
authors: Krieger
authors: HAS-UD Vascular Biology, Thrombosis and Hemostasis Research Group, Hungarian Academy of Sciences, Debrecen, Hungary Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors: István
authors: I
authors: Komáromi
authors: German Cancer Research Center (DKFZ), Biophysics of Macromolecules, Heidelberg, Germany.
authors: Katalin
authors: K
authors: Tóth
authors: Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary Sanford Burnham Prebys Medical Discovery Institute at Lake Nona, Orlando, Florida, USA vamosig@med.unideb.hu.
authors: György
authors: G
authors: Vámosi
mesh_terms: D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D055503:Protein Multimerization; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000032:analysis / Q000378:metabolism
chemical_list: D002843:Chromatin; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D004247:DNA