Rivera-Casas Ciro, Gonzalez-Romero Rodrigo, Garduño Rafael A, Cheema Manjinder S, Ausio Juan, Eirin-Lopez Jose M
Environmental Epigenetics Group, Department of Biological Sciences, Florida International UniversityNorth Miami, FL, United States.
Department of Microbiology and Immunology, Dalhousie UniversityHalifax, NS, Canada.
Front Physiol. 2017 Aug 8;8:490. doi: 10.3389/fphys.2017.00490. eCollection 2017.
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.
双壳贝类是一个广泛存在的分类群体，在几乎所有生态系统中都发挥着关键作用，并且具有重要的商业价值。在大多数情况下，双壳贝类具有固着和滤食的生活方式，这些特性使其成为常用于水生栖息地环境监测研究的典型指示生物。鉴于表观遗传修饰在适应和适应过程中的作用，研究将环境暴露与特定生理反应联系起来的表观遗传机制（即环境表观遗传学）是一种非常创新的监测策略。此外，许多此类修饰的可遗传性质为探索表观遗传调控和选择在管理和恢复策略中的适用性提供了一条非常有前景的途径。染色质为研究环境表观遗传反应提供了一个框架。不幸的是，在大多数非传统模式生物中，染色质和表观遗传信息非常有限，甚至在大多数与环境和生态相关的生物中完全缺乏。本研究旨在为双壳贝类染色质的研究提供一个全面且可重复的实验工作流程。首先，提供了一系列用于编码与染色质相关蛋白质的基因的分子分离指南，包括适用于常规PCR、cDNA末端快速扩增（RACE）、基因组步移和定量PCR（qPCR）实验的引物信息。本节之后描述了专门为分析不同双壳贝类组织中的组蛋白和SNBP蛋白而开发的方法，包括蛋白质提取、纯化、分离和免疫检测。最后，还提供了有关可用抗体及其特异性和性能的信息。这里描述的工具和方案通过纳入对调节染色质动态的结构元件的研究，补充了当前的表观遗传分析（通常限于DNA甲基化）。
