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Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay

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      Abstract

      Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein–DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, BrU-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.

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      A census of human transcription factors: function, expression and evolution.

      Transcription factors are key cellular components that control gene expression: their activities determine how cells function and respond to the environment. Currently, there is great interest in research into human transcriptional regulation. However, surprisingly little is known about these regulators themselves. For example, how many transcription factors does the human genome contain? How are they expressed in different tissues? Are they evolutionarily conserved? Here, we present an analysis of 1,391 manually curated sequence-specific DNA-binding transcription factors, their functions, genomic organization and evolutionary conservation. Much remains to be explored, but this study provides a solid foundation for future investigations to elucidate regulatory mechanisms underlying diverse mammalian biological processes.
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        Transcription regulation and animal diversity.

        Whole-genome sequence assemblies are now available for seven different animals, including nematode worms, mice and humans. Comparative genome analyses reveal a surprising constancy in genetic content: vertebrate genomes have only about twice the number of genes that invertebrate genomes have, and the increase is primarily due to the duplication of existing genes rather than the invention of new ones. How, then, has evolutionary diversity arisen? Emerging evidence suggests that organismal complexity arises from progressively more elaborate regulation of gene expression.
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          Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors.

           Aziz Sancar (2003)
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            Author and article information

            Affiliations
            [1 ]Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan
            [2 ]Department of Molecular Cell Biology, Sungkyunkwan University, School of Medicine, Suwon 440-746, Korea
            [3 ]Institute for Integrated Cell-Materials Sciences (iCeMS) Kyoto University, Yoshida-ushinomiyacho, Sakyo-Ku, Kyoto 606-8501, Japan
            Author notes
            [* ]To whom correspondence should be addressed. Tel: +81 75 753 4002; Fax: +81 75 753 3670; Email: hs@ 123456kuchem.kyoto-u.ac.jp
            Correspondence may also be addressed to Kyeong Kyu Kim. Tel: +82 31 299 6136; Fax: +82 31 299 6269; Email: kyeongkyu@ 123456skku.edu
            Journal
            Nucleic Acids Res
            Nucleic Acids Res
            nar
            nar
            Nucleic Acids Research
            Oxford University Press
            0305-1048
            1362-4962
            19 August 2016
            26 May 2016
            26 May 2016
            : 44
            : 14
            : e125
            27229137
            5001601
            10.1093/nar/gkw478
            © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

            This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

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            Pages: 9
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            16
            Methods Online
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            19 August 2016

            Genetics

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