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.
Highlights
Epigenetics and the Structure of ChromatinIn eukaryotes, DNA is packaged and compacted within the cell nucleus thanks to its association with chromosomal proteins, constituting the chromatin fiber
Higher-order chromatin structures are formed by the incorporation of linker histones (H1 family) which bind to adjacent nucleosomes and linker-DNA, facilitating the compaction of the chromatin fiber (Simpson, 1978)
Environmental epigenetics is at the center stage of such efforts, given the role of epigenetic modifications during the regulation of gene activity, and their implications for acclimatization and adaptation under ever-changing environments
Summary
Epigenetics and the Structure of ChromatinIn eukaryotes, DNA is packaged and compacted within the cell nucleus thanks to its association with chromosomal proteins, constituting the chromatin fiber. The dynamic exchange of histones in nucleosomes genome-wide may result in heritable (i.e., epigenetic) alterations in chromatin structure, regulating the accessibility of DNA for transcription, replication and repair factors involved in DNA metabolism (Wang et al, 2007b). There, histones are almost completely replaced by smaller and even more basic proteins known as Sperm Nuclear Basic Proteins (SNBPs) (Ausio et al, 2007). These proteins can be divided into three types, evolutionary related to the linker histone H1, including: histone (H) type, protamine-like (PL) type, and protamine (P) type (Eirin-Lopez and Ausio, 2009). The study of germ chromatin structure and function is indispensable to understand how epigenetic marks are trans-generationally inherited
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