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.
Highlights
Natural environments generate complex, often noisy, natura, are crucial for validating gene regulatory mechanisms defined under laboratory conditions and are, important for addressing novel questions that are specific to natural conditions (Kudoh and Nagano, 2013; Kudoh, 2015)
While the basis of this mechanism remained largely unknown, a wealth of molecular data available from laboratory studies of Arabidopsis thaliana shows that A. thaliana FLOWERING LOCUS C (FLC) is regulated by a universal cellular memory machinery based on two major protein complexes with antagonistic activities: Trithorax complex catalyses the trimethylation of lysine 4 of histone H3 tails (H3K4me3), which is involved in maintenance of active gene transcription, while Polycomb complex catalyses the trimethylation of lysine 27 of histone H3 tails (H3K27me3), which is involved in maintenance of repressed gene activity (Buzas et al, 2012; Song et al, 2012)
While these constant, simplified conditions greatly facilitated the initial dissection of molecular mechanisms of gene expression (e.g., Michaels and Amasino, 1999) and histone modifications (e.g., Bastow et al, 2004; Sung and Amasino, 2004) during vernalization, such simplification may not be informative about molecular mechanisms operating under fluctuating natural environments
Summary
Often noisy, natura, are crucial for validating gene regulatory mechanisms defined under laboratory conditions and are, important for addressing novel questions that are specific to natural conditions (Kudoh and Nagano, 2013; Kudoh, 2015). A comprehensive in natura gene expression profile of the central gene of this process, FLOWERING LOCUS C (FLC), was reported for a two-year complete seasonal cycle of Arabidopsis halleri subsp. Gemmifera (A. halleri, hereafter) (Aikawa et al, 2010) Those quantitative gene expression data demonstrated that A. halleri FLC (AhgFLC) expression follows the seasonal trend of the temperature. Modelling of the gene expression data versus environmental temperature led to the conclusion that the seasonal gene expression was controlled by temperatures in the preceding six weeks, which in turn led the authors to postulate the existence of a long-term memory mechanism that functions to filter out short-term noise (Aikawa et al, 2010). While the basis of this mechanism remained largely unknown, a wealth of molecular data available from laboratory studies of Arabidopsis thaliana shows that A. thaliana FLC is regulated by a universal cellular memory machinery based on two major protein complexes with antagonistic activities: Trithorax complex catalyses the trimethylation of lysine 4 of histone H3 tails (H3K4me3), which is involved in maintenance of active gene transcription, while Polycomb complex catalyses the trimethylation of lysine 27 of histone H3 tails (H3K27me3), which is involved in maintenance of repressed gene activity (Buzas et al, 2012; Song et al, 2012)
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