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From the laboratory to the field: assaying histone methylation at <i>FLOWERING LOCUS C</i> in naturally growing <i>Arabidopsis halleri</i>

  • Nishio Haruki
    Center for Ecological Research, Kyoto University
  • Buzas Diana Mihaela
    Gene Research Center, Faculty of Life and Environmental Sciences, University of Tsukuba
  • Nagano Atsushi J.
    Center for Ecological Research, Kyoto University Japan Science and Technology Agency, PRESTO Department of Plant Life Sciences, Faculty of Agriculture, Ryukoku University
  • Suzuki Yutaka
    Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo
  • Sugano Sumio
    Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo
  • Ito Motomi
    Department of General Systems Studies, Graduate School of Arts and Sciences, University of Tokyo
  • Morinaga Shin-Ichi
    College of Bioresource Sciences, Nihon University Japan Science and Technology Agency, CREST
  • Kudoh Hiroshi
    Center for Ecological Research, Kyoto University

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  • From the laboratory to the field : assaying histone methylation at FLOWERING LOCUS C in naturally growing Arabidopsis halleri

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Abstract

Gene regulatory mechanisms are often defined in studies performed in the laboratory but are seldom validated for natural habitat conditions, i.e., in natura. Vernalization, the promotion of flowering by winter cold, is a prominent naturally occurring phenomenon, so far best characterized using artificial warm and cold treatments. The floral inhibitor FLOWERING LOCUS C (FLC) gene of Arabidopsis thaliana has been identified as the central regulator of vernalization. FLC shows an idiosyncratic pattern of histone modification at different stages of cold exposure, believed to regulate transcriptional responses of FLC. Chromatin modifications, including H3K4me3 and H3K27me3, are routinely quantified using chromatin immunoprecipitation (ChIP), standardized for laboratory samples. In this report, we modified a ChIP protocol to make it suitable for analysis of field samples. We first validated candidate normalization control genes at two stages of cold exposure in the laboratory and two seasons in the field, also taking into account nucleosome density. We further describe experimental conditions for performing sampling and sample preservation in the field and demonstrate that these conditions give robust results, comparable with those from laboratory samples. The ChIP protocol incorporating these modifications, “Field ChIP”, was used to initiate in natura chromatin analysis of AhgFLC, an FLC orthologue in A. halleri, of which a natural population is already under investigation. Here, we report results on levels of H3K4me3 and H3K27me3 at three representative regions of AhgFLC in controlled cold and field samples, before and during cold exposure. We directly compared the results in the field with those from laboratory samples. These data revealed largely similar trends in histone modification dynamics between laboratory and field samples at AhgFLC, but also identified some possible differences. The Field ChIP method described here will facilitate comprehensive chromatin analysis of AhgFLC in the future to contribute to our understanding of gene regulation in fluctuating natural environments.

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