Histonas variantes en invertebrados marinos: puesta a punto de metodologías para el estudio de la cromatina y caracterización de las variantes macroH2A y H2A.Z.2 en moluscos bivalvos

[Resumen] En las células eucariotas el DNA se asocia con histonas y proteínas no histónicas formando un complejo conocido como cromatina que media en la organización y la regulación del material hereditario en el espacio del núcleo celular. Además del papel estructural de las histonas, la incorporación de variantes de estas proteínas a los nucleosomas es esencial para el correcto desarrollo de importantes procesos celulares como la regulación de la expresión génica, la replicación o la reparación del DNA. Aunque existe una gran cantidad de estudios sobre estas proteínas en organismos deuteróstomos, la presencia y características funcionales de histonas variantes, además de la especialización que imparten a la cromatina, son prácticamente desconocidas en el caso del linaje de animales protóstomos. Dentro de este grupo, los moluscos bivalvos presentan un interés especial debido a las condiciones ambientales a las que están expuestos que suscita una rápida y precisa regulación de la expresión génica. La caracterización de las histonas variantes macroH2A y H2A.Z.2 en especies de este grupo animal, así como el desarrollo de técnicas de estudio de su cromatina, aporta datos muy valiosos acerca de la especialización funcional impartida por estas proteínas a la cromatina de los diversos grupos de organismos eucariotas.[Resumo] Nas células eucarióticas o DNA asóciase con histonas e proteínas non histónicas formando un complexo coñecido como cromatina que media na organización e a regulación do material hereditario no espazo do núcleo celular. Ademais do papel estrutural das histonas, a incorporación de variantes destas proteínas aos nucleosomas é esencial para o correcto desenvolvemento de importantes procesos celulares como a regulación da expresión xénica, a replicación ou a reparación do DNA. Aínda que existe unha gran cantidade de estudos sobre estas proteínas en organismos deuteróstomos, a presenza e características funcionais de histonas variantes, ademais da especialización que imparten á cromatina, son practicamente descoñecidas no caso da liñaxe dos animais protóstomos. Dentro deste grupo, os moluscos bivalvos presentan un interese especial debido ás condicións ambientais ás que están expostos que suscita unha rápida e precisa regulación da expresión xénica. A caracterización das histonas variantes macroH2A e H2A.Z.2 en especies deste grupo animal, así como o desenvolvemento de técnicas de estudo da súa cromatina, achega datos moi valiosos acerca da especialización funcional impartida por estas proteínas á cromatina dos diversos grupos de organismos eucarióticas[Abstract] In eukaryotic cells DNA is associated with histone and non-histone proteins constituting a nucleoprotein structure known as chromatin, mediating the organization and regulation of the genetic material in the cell nucleus. Besides the structural role of histones, incorporation of variants of these proteins to nucleosomes is essential for the proper development of important cellular processes such as regulation of gene expression, replication, and DNA repair. Although several studies on these proteins are available in deuterostome organisms, the presence and functional characteristics of histone variants and the specialized functions imparted to the chromatin are virtually unknown in the case of the protostome lineage. Within this group, bivalve molluscs are of particular interest because of the environmental conditions to which they are exposed. The characterization of histone variants macroH2A and H2A.Z.2 in these organisms and the development of different techniques for studying their chromatin will provide valuable information about the functional specialization imparted by these proteins to the chromatin of eukaryotic organisms

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

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

Characterization of mussel H2A.Z.2: a new H2A.Z variant preferentially expressed in germinal tissues from Mytilus

Histones are the fundamental constituents of the eukaryotic chromatin, facilitating the physical organization of DNA in chromosomes and participating in the regulation of its metabolism. The H2A family displays the largest number of variants among core histones, including the renowned H2A.X, macroH2A, H2A.B (Bbd) and H2A.Z. This latter variant is especially interesting due to its regulatory role and its differentiation into two functionally divergent variants (H2A.Z.1 and H2A.Z.2), further specializing the structure and function of vertebrate chromatin. In the present work we describe, for the first time, the presence of a second H2A.Z variant (H2A.Z.2) in the genome of a non-vertebrate animal, the mussel Mytilus. The molecular and evolutionary characterization of mussel H2A.Z.1 and H2A.Z.2 histones is consistent with their functional specialization, supported on sequence divergence at promoter and coding regions as well as on varying gene expression patterns. More precisely, the expression of H2A.Z.2 transcripts in gonadal tissue and its potential upregulation in response to genotoxic stress might be mirroring the specialization of this variant in DNA repair. Overall, the findings presented in this work complement recent reports describing the widespread presence of other histone variants across eukaryotes, supporting an ancestral origin and conserved role for histone variants in chromatin

The CHROMEVALOA Database: A Resource for the Evaluation of Okadaic Acid Contamination in the Marine Environment Based on the Chromatin-Associated Transcriptome of the Mussel Mytilus galloprovincialis

Okadaic Acid (OA) constitutes the main active principle in Diarrhetic Shellfish Poisoning (DSP) toxins produced during Harmful Algal Blooms (HABs), representing a serious threat for human consumers of edible shellfish. Furthermore, OA conveys critical deleterious effects for marine organisms due to its genotoxic potential. Many efforts have been dedicated to OA biomonitoring during the last three decades. However, it is only now with the current availability of detailed molecular information on DNA organization and the mechanisms involved in the maintenance of genome integrity, that a new arena starts opening up for the study of OA contamination. In the present work we address the links between OA genotoxicity and chromatin by combining Next Generation Sequencing (NGS) technologies and bioinformatics. To this end, we introduce CHROMEVALOAdb, a public database containing the chromatin-associated transcriptome of the mussel Mytilus galloprovincialis (a sentinel model organism) in response to OA exposure. This resource constitutes a leap forward for the development of chromatin-based biomarkers, paving the road towards the generation of powerful and sensitive tests for the detection and evaluation of the genotoxic effects of OA in coastal areas

Histone H2A (H2A.X and H2A.Z) Variants in Molluscs: Molecular Characterization and Potential Implications For Chromatin Dynamics

Histone variants are used by the cell to build specialized nucleosomes, replacing canonical histones and generating functionally specialized chromatin domains. Among many other processes, the specialization imparted by histone H2A (H2A.X and H2A.Z) variants to the nucleosome core particle constitutes the earliest response to DNA damage in the cell. Consequently, chromatin-based genotoxicity tests have been developed in those cases where enough information pertaining chromatin structure and dynamics is available (i.e., human and mouse). However, detailed chromatin knowledge is almost absent in most organisms, specially protostome animals. Molluscs (which represent sentinel organisms for the study of pollution) are not an exception to this lack of knowledge. In the present work we first identified the existence of functionally differentiated histone H2A.X and H2A.Z variants in the mussel Mytilus galloprovincialis (MgH2A.X and MgH2A.Z), a marine organism widely used in biomonitoring programs. Our results support the functional specialization of these variants based on: a) their active expression in different tissues, as revealed by the isolation of native MgH2A.X and MgH2A.Z proteins in gonad and hepatopancreas; b) the evolutionary conservation of different residues encompassing functional relevance; and c) their ability to confer specialization to nucleosomes, as revealed by nucleosome reconstitution experiments using recombinant MgH2A.X and MgH2A.Z histones. Given the seminal role of these variants in maintaining genomic integrity and regulating gene expression, their preliminary characterization opens up new potential applications for the future development of chromatin-based genotoxicity tests in pollution biomonitoring programs

The characterization of macroH2A beyond vertebrates supports an ancestral origin and conserved role for histone variants in chromatin

Histone variants play a critical role in chromatin structure and epigenetic regulation. These “deviant” proteins have been historically considered as the evolutionary descendants of ancestral canonical histones, helping specialize the nucleosome structure during eukaryotic evolution. Such view is now challenged by 2 major observations: first, canonical histones present extremely unique features not shared with any other genes; second, histone variants are widespread across many eukaryotic groups. The present work further supports the ancestral nature of histone variants by providing the first in vivo characterization of a functional macroH2A histone (a variant long defined as a specific refinement of vertebrate chromatin) in a non-vertebrate organism (the mussel Mytilus) revealing its recruitment into heterochromatic fractions of actively proliferating tissues. Combined with in silico analyses of genomic data, these results provide evidence for the widespread presence of macroH2A in metazoan animals, as well as in the holozoan Capsaspora, supporting an evolutionary origin for this histone variant lineage before the radiation of Filozoans (including Filasterea, Choanoflagellata and Metazoa). Overall, the results presented in this work help configure a new evolutionary scenario in which histone variants, rather than modern “deviants” of canonical histones, would constitute ancient components of eukaryotic chromatin