Molecular and Biochemical Methods Useful for the Epigenetic Characterization of Chromatin-Associated Proteins in Bivalve Molluscs

Abstract

Bivalve molluscs constitute a ubiquitous taxonomic group playing key functions in virtually all ecosystems, and encompassing critical commercial relevance. Along with a sessile and filter-feeding lifestyle in most cases, these characteristics make bivalves model sentinel organisms routinely used for environmental monitoring studies in aquatic habitats. The study of epigenetic mechanisms linking environmental exposure and specific physiological responses (i.e., environmental epigenetics) stands out as a very innovative monitoring strategy, given the role of epigenetic modifications in acclimatization and adaptation. Furthermore, the heritable nature of many of those modifications constitutes a very promising avenue to explore the applicability of epigenetic conditioning and selection in management and restoration strategies. Chromatin provides a framework for the study of environmental epigenetic responses. Unfortunately, chromatin and epigenetic information are very limited in most non-traditional model organisms and even completely lacking in most environmentally and ecologically relevant organisms. The present work aims to provide a comprehensive and reproducible experimental workflow for the study of bivalve chromatin. First, a series of guidelines for the molecular isolation of genes encoding chromatin-associated proteins is provided, including information on primers suitable for conventional PCR, Rapid Amplification of cDNA Ends (RACE), genome walking and quantitative PCR (qPCR) experiments. This section is followed by the description of methods specifically developed for the analysis of histone and SNBP proteins in different bivalve tissues, including protein extraction, purification, separation and immunodetection. Lastly, information about available antibodies, their specificity and performance is also provided. The tools and protocols described here complement current epigenetic analyses (usually limited to DNA methylation) by incorporating the study of structural elements modulating chromatin dynamics.

Coral epigenetic responses to nutrient stress: Histone H2A.X phosphorylation dynamics and DNA methylation in the staghorn coral Acropora cervicornis

“…Epigenetic modifications in invertebrates can occur rapidly after exposure to environmental stress (Gonzalez‐Romero et al, ; Rivera‐Casas et al, ; Suarez‐Ulloa, Gonzalez‐Romero, & Eirin‐Lopez, ). Therefore, coral fragments were sampled at three different times during day 1 of exposure (1, 2, and 5 hr), day 2, day 7, and weekly thereafter for the next 4 weeks.…”

“…Histone proteins re isolated as described elsewhere and adapted to coral tissue in the present work (Rivera‐Casas et al, ). Accordingly, 5 mg of holobiont tissue were homogenized in a buffer consisting of 100 mM KCl, 50 mM Tris‐HCl, 1 Mm MgCl 2%, and 0.5% Triton X‐100 (pH 7.5) and containing a protease inhibitor mixture.…”

“…Gels were stained with 0.2% (w/v) Coomassie blue in 25% (v/v) 2‐propanol, 10% (v/v) acetic acid and de‐stained in 10% (v/v) 2‐propanol, 10% (v/v) acetic acid. Additional histone separation was carried out using high‐performance liquid chromatography (HPLC) as described in Rivera‐Casas et al (). Histone proteins were detected using commercial antibodies in western blot analyses, including anti‐H2A.X (H2A.X.ab, Abcam Cambridge, MA; H2A.Xry; Raybiotech, Norcross, GA) and anti‐γH2A.X (γ‐H2A.X ab, Rockland, Pottstown, PA; γ‐H2A.Xry, Raybiotech).…”

“…Histone proteins re isolated as described elsewhere and adapted to coral tissue in the present work (Rivera-Casas et al, 2017).…”

“…mance liquid chromatography (HPLC) as described inRivera-Casas et al (2017). Histone proteins were detected using commercial antibodies in western blot analyses, including anti-H2A.X (H2A.X.ab, Abcam Cambridge, MA; H2A.Xry; Raybiotech, Norcross, GA) and anti-γH2A.X (γ-H2A.X ab, Rockland, Pottstown, PA; γ-H2A.Xry, Raybiotech).…”