RESUMEN
Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing. Here, we primarily used electron transfer dissociation MS-based analyses, revealing that the protamines in the sperm of the liverwort Marchantia polymorpha result from post-translational cleavage of three precursor H1 histones. Moreover, we show that the mature protamines are further post-translationally modified by di-aminopropanelation, and previous studies have reported that they condense spermatid chromatin through a process consisting of liquid-phase assembly likely involving spinodal decomposition. Taken together, our results reveal that the interesting evolutionary ancestry of protamines begins with histone H1 in both the animal and plant kingdoms.
Asunto(s)
Marchantia/metabolismo , Protaminas/metabolismo , Secuencia de Aminoácidos/genética , Animales , Cromatina/metabolismo , Hepatophyta/metabolismo , Histonas/metabolismo , Masculino , Espectrometría de Masas/métodos , Protaminas/genética , Procesamiento Proteico-Postraduccional/fisiología , Espermátides/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismoRESUMEN
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 because of its regulatory role and its differentiation into 2 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.
Asunto(s)
Centro Germinal/metabolismo , Mytilus/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Histonas/metabolismo , Mutación/genética , Mytilus/genética , Filogenia , Conformación Proteica , Proteínas/química , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Bases de Datos Factuales , Mutágenos/análisis , Mytilus/genética , Ácido Ocadaico/análisis , Animales , Carcinógenos/análisis , Carcinógenos/aislamiento & purificación , Carcinógenos/toxicidad , Cromatina/metabolismo , Monitoreo del Ambiente/métodos , Humanos , Pruebas de Mutagenicidad/métodos , Mutágenos/aislamiento & purificación , Mutágenos/toxicidad , Ácido Ocadaico/toxicidad , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
The rich diversity within each of the five histone families (H1, H2A, H2B, H3, and H4) can hardly be reconciled with the notion of homogenizing evolution. The prevalence of birth-and-death long-term evolution over concerted evolution has already been demonstrated in the linker histone H1 family as well as for the H2A, H3, and H4 core histone families. However, information about histone H2B is lacking. In the present work, we have analyzed the diversity of the members of this histone family across different eukaryotic genomes and have characterized the mechanisms involved in their long-term evolution. Our results reveal that, quite in contrast with other histones, H2B variants are subject to a very rapid process of diversification that primarily affects the male germinal cell lineage and involves their functional specialization probably as a consequence of neofunctionalization and subfunctionalization events after gene duplication. The overall parallelism observed between the molecular phylogenies and the relationships among the electrostatic potentials of the different variants suggests that the latter may have played a major structural selective constraint during H2B evolution. It thus seems that the reorganization of chromatin structure during spermiogenesis might have affected the evolutionary constraints driving histone H2B evolution, leading to an increase in diversity.
Asunto(s)
Evolución Molecular , Células Germinativas/fisiología , Histonas/genética , Isoformas de Proteínas/genética , Animales , Linaje de la Célula , Bases de Datos Genéticas , Eucariontes/genética , Duplicación de Gen , Genoma , Células Germinativas/citología , Histonas/química , Histonas/clasificación , Humanos , Masculino , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/clasificación , Estructura Terciaria de Proteína , Seudogenes , Electricidad EstáticaRESUMEN
NAD metabolism is essential for all forms of life. Compartmental regulation of NAD+ consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD+ consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.
Asunto(s)
Metabolismo Energético/fisiología , Histonas/genética , Histonas/metabolismo , NAD/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Reparación del ADN/genética , Eucariontes/metabolismo , Humanos , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidoresRESUMEN
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.
RESUMEN
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.
Asunto(s)
Cromatina/genética , Evolución Molecular , Histonas/genética , Animales , Cromatina/metabolismo , Secuencia Conservada , Código de Histonas , Histonas/metabolismo , Anfioxos/genética , Ratones , Mytilus/genética , Anémonas de Mar/genética , Garrapatas/genéticaRESUMEN
Marine biotoxins synthesized by Harmful Algal Blooms (HABs) represent one of the most important sources of contamination in marine environments as well as a serious threat to fisheries and aquaculture-based industries in coastal areas. Among these biotoxins Okadaic Acid (OA) is of critical interest as it represents the most predominant Diarrhetic Shellfish Poisoning biotoxin in the European coasts. Furthermore, OA is a potent tumor promoter with aneugenic and clastogenic effects on the hereditary material, most notably DNA breaks and alterations in DNA repair mechanisms. Therefore, a great effort has been devoted to the biomonitoring of OA in the marine environment during the last two decades, mainly based on physicochemical and physiological parameters using mussels as sentinel organisms. However, the molecular genotoxic effects of this biotoxin make chromatin structure a good candidate for an alternative strategy for toxicity assessment with faster and more sensitive evaluation. To date, the development of chromatin-based studies to this purpose has been hampered by the complete lack of information on chromatin of invertebrate marine organisms, especially in bivalve molluscs. Our preliminary results have revealed the presence of histone variants involved in DNA repair and chromatin specialization in mussels and clams. In this work we use this information to put forward a proposal focused on the development of chromatin-based tests for OA genotoxicity in the marine environment. The implementation of such tests in natural populations has the potential to provide an important leap in the biomonitoring of this biotoxin. The outcome of such monitoring may have critical implications for the evaluation of DNA damage in these marine organisms. They will provide as well important tools for the optimization of their harvesting and for the elaboration of additional tests designed to evaluate the safety of their consumption and potential implications for consumer's health.
Asunto(s)
Bivalvos/efectos de los fármacos , Cromatina/efectos de los fármacos , Pruebas de Mutagenicidad/métodos , Ácido Ocadaico/toxicidad , Animales , Bivalvos/clasificación , Bivalvos/genética , Carcinógenos Ambientales/toxicidad , Cromatina/genética , Daño del ADN , Monitoreo del Ambiente/métodos , Toxinas Marinas/toxicidad , Especificidad de la EspecieRESUMEN
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.