In vivo and in vitro footprinting of nucleosomes and transcriptional activators using an infrared-fluorescence DNA sequencer

Nobuyuki Morohashi, Kumiko Nakajima, Shunsuke Kuwana, Hiroaki Tachiwana, Hitoshi Kurumizaka, Mitsuhiro Shimizu

    Research output: Contribution to journalArticle

    3 Citations (Scopus)

    Abstract

    The analysis of nucleosome positions and transcription factor binding in chromatin is a central issue for understanding the mechanisms of gene expression in eukaryotes. Here, we have developed a footprinting technique, using multi-cycle primer extension with an infrared-fluorescence DNA sequencer, to analyze chromatin structure in isolated yeast nuclei and transcriptional activator binding in living yeast cells. Using this technique, the binding of the yeast activators Hap1 and Hap2/3/4/5 to their cognate sites was detectable as hypersensitive sites by in vivo UV-photofootprinting, and the locations of nucleosomes in yeast minichromosomes were determined by micrococcal nuclease mapping. We also applied this method to determine the position of the nucleosome in the 5S DNA fragment reconstituted in vitro. This technique allowed us to eliminate the use of radioactive materials and to perform experiments on common benches. Thus, the footprinting procedure established in this study will be useful to researchers studying DNA-protein interactions and chromatin structure in vivo and in vitro.

    Original languageEnglish
    Pages (from-to)187-192
    Number of pages6
    JournalBiological and Pharmaceutical Bulletin
    Volume31
    Issue number2
    DOIs
    Publication statusPublished - 2008 Feb

    Fingerprint

    Nucleosomes
    Yeasts
    Fluorescence
    Chromatin
    DNA
    Micrococcal Nuclease
    Eukaryota
    Transcription Factors
    Research Personnel
    Gene Expression
    In Vitro Techniques
    Proteins

    Keywords

    • Chromatin
    • DNA footprinting
    • DNA sequencer
    • DNA-protein interaction
    • Nucleosome
    • Transcription factor

    ASJC Scopus subject areas

    • Molecular Medicine
    • Pharmacology, Toxicology and Pharmaceutics(all)

    Cite this

    In vivo and in vitro footprinting of nucleosomes and transcriptional activators using an infrared-fluorescence DNA sequencer. / Morohashi, Nobuyuki; Nakajima, Kumiko; Kuwana, Shunsuke; Tachiwana, Hiroaki; Kurumizaka, Hitoshi; Shimizu, Mitsuhiro.

    In: Biological and Pharmaceutical Bulletin, Vol. 31, No. 2, 02.2008, p. 187-192.

    Research output: Contribution to journalArticle

    Morohashi, Nobuyuki ; Nakajima, Kumiko ; Kuwana, Shunsuke ; Tachiwana, Hiroaki ; Kurumizaka, Hitoshi ; Shimizu, Mitsuhiro. / In vivo and in vitro footprinting of nucleosomes and transcriptional activators using an infrared-fluorescence DNA sequencer. In: Biological and Pharmaceutical Bulletin. 2008 ; Vol. 31, No. 2. pp. 187-192.
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    AU - Tachiwana, Hiroaki

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