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      Mechanisms of Unphosphorylated STAT3 Transcription Factor Binding to DNA*

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          Abstract

          Background: Unphosphorylated STAT3 (U-STAT3) regulates gene expression, but the mechanisms of its DNA binding are not fully understood.

          Results: U-STAT3 binds to the same γ-activated sequence (GAS) DNA-binding site as phosphorylated STAT3. It also binds to AT-rich DNA structures.

          Conclusion: U-STAT3 regulates gene expression by binding to GAS and influencing chromatin organization.

          Significance: Our data provide an explanation of mechanisms of U-STAT3 binding to DNA.

          Abstract

          Phosphorylation of signal transducer and activator of transcription 3 (STAT3) on a single tyrosine residue in response to growth factors, cytokines, interferons, and oncogenes activates its dimerization, translocation to the nucleus, binding to the interferon γ ( gamma)- activated sequence (GAS) DNA-binding site and activation of transcription of target genes. STAT3 is constitutively phosphorylated in various cancers and drives gene expression from GAS-containing promoters to promote tumorigenesis. Recently, roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in maintenance of heterochromatin stability. However, the mechanisms underlying U-STAT3 binding to DNA has not been fully investigated. Here, we explore STAT3-DNA interactions by atomic force microscopy (AFM) imaging. We observed that U-STAT3 molecules bind to the GAS DNA-binding site as dimers and monomers. In addition, we observed that U-STAT3 binds to AT-rich DNA sequence sites and recognizes specific DNA structures, such as 4-way junctions and DNA nodes, within negatively supercoiled plasmid DNA. These structures are important for chromatin organization and our data suggest a role for U-STAT3 as a chromatin/genome organizer. Unexpectedly, we found that a C-terminal truncated 67.5-kDa STAT3 isoform recognizes single-stranded spacers within cruciform structures that also have a role in chromatin organization and gene expression. This isoform appears to be abundant in the nuclei of cancer cells and, therefore, may have a role in regulation of gene expression. Taken together, our data highlight novel mechanisms by which U-STAT3 binds to DNA and supports U-STAT3 function as a transcriptional activator and a chromatin/genomic organizer.

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          Most cited references46

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          Stat3 as an oncogene.

          STATs are latent transcription factors that mediate cytokine- and growth factor-directed transcription. In many human cancers and transformed cell lines, Stat3 is persistently activated, and in cell culture, active Stat3 is either required for transformation, enhances transformation, or blocks apoptosis. We report that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors.
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            Protein-binding assays in biological liquids using microscale thermophoresis.

            Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.
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              Molecular interaction studies using microscale thermophoresis.

              Abstract The use of infrared laser sources for creation of localized temperature fields has opened new possibilities for basic research and drug discovery. A recently developed technology, Microscale Thermophoresis (MST), uses this temperature field to perform biomolecular interaction studies. Thermophoresis, the motion of molecules in temperature fields, is very sensitive to changes in size, charge, and solvation shell of a molecule and thus suited for bioanalytics. This review focuses on the theoretical background of MST and gives a detailed overview on various applications to demonstrate the broad applicability. Experiments range from the quantification of the affinity of low-molecular-weight binders using fluorescently labeled proteins, to interactions between macromolecules and multi-component complexes like receptor containing liposomes. Information regarding experiment and experimental setup is based on the Monolith NT.115 instrument (NanoTemper Technologies GmbH).
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                20 April 2012
                29 February 2012
                29 February 2012
                : 287
                : 17
                : 14192-14200
                Affiliations
                From the Departments of []Oncology,
                [§ ]Radiation Medicine,
                []Neuroscience,
                [‡‡ ]Biostatistics, Bioinformatics, and Biomathematics, and
                [§§ ]Drug Discovery Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057 and
                the []Cancer and Inflammation Program,
                [** ]Biophysics Resource Core, Structural Biophysics Laboratory, NCI-Frederick, Frederick, Maryland 21702
                Author notes
                [2 ] To whom correspondence should be addressed: Dept. of Oncology, Research Building, R. E216, 3970 Reservoir Rd., NW, Washington, D. C. 20057-1482. Tel.: 202-687-8810; Fax: 202-687-2221; E-mail: oat@ 123456georgetown.edu .
                [1]

                Both authors contributed equally.

                Article
                M111.323899
                10.1074/jbc.M111.323899
                3340179
                22378781
                6203e6ab-48af-4d2a-a49a-633877d91025
                © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 15 November 2011
                : 27 February 2012
                Funding
                Funded by: National Institutes of Health
                Award ID: CA56036-08
                Categories
                Gene Regulation

                Biochemistry
                stat3,dna binding,stat transcription factor,microscale thermophoresis,transcription factors,dna structure,atomic force microscopy

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