The protamines of the noble false widow spider Steatoda nobilis provide an example of liquid-liquid phase separation chromatin transitions during spermiogenesis.

While there is extensive information about sperm nuclear basic proteins (SNBP) in vertebrates, there is very little information about Arthropoda by comparison. This paper aims to contribute to filling this gap by analyzing these proteins in the sperm of the noble false widow spider Steatoda nobilis (Order Araneae, Family Theridiidae). To this end, we have developed a protein extraction method that allows the extraction of cysteine-containing protamines suitable for the preparation and analysis of SNBPs from samples where the amount of starting tissue material is limited. We carried out top-down mass spectrometry sequencing and molecular phylogenetic analyses to characterize the protamines of S. nobilis and other spiders. We also used electron microscopy to analyze the chromatin organization of the sperm, and we found it to exhibit liquid-liquid phase spinodal decomposition during the late stages of spermiogenesis. These studies further our knowledge of the distribution of SNBPs within the animal kingdom and provide additional support for a proposed evolutionary origin of many protamines from a histone H1 (H5) replication-independent precursor.

A complex interplay between H2A.Z and HP1 isoforms regulates pericentric heterochromatin.

Pericentric heterochromatin (PCH) plays an essential role in the maintenance of genome integrity and alterations in PCH have been linked to cancer and aging. HP1 α, ß, and γ, are hallmarks of constitutive heterochromatin that are thought to promote PCH structure through binding to heterochromatin-specific histone modifications and interaction with a wide range of factors. Among the less understood components of PCH is the histone H2A variant H2A.Z, whose role in the organization and maintenance of PCH is poorly defined. Here we show that there is a complex interplay between H2A.Z and HP1 isoforms in PCH. While the loss of HP1α results in the accumulation of H2A.Z.1 in PCH, which is associated with a significant decrease in its mobile fraction, H2A.Z.1 binds preferentially to HP1ß in these regions. Of note, H2A.Z.1 downregulation results in increased heterochromatinization and instability of PCH, reflected by accumulation of the major epigenetic hallmarks of heterochromatin in these regions and increased frequency of chromosome aberrations related to centromeric/pericentromeric defects. Our studies support a role for H2A.Z in genome stability and unveil a key role of H2A.Z in the regulation of heterochromatin-specific epigenetic modifications through a complex interplay with the HP1 isoforms.

Chromatin gatekeeper and modifier CHD proteins in development, and in autism and other neurological disorders.

Chromatin, a protein-DNA complex, is a dynamic structure that stores genetic information within the nucleus and responds to molecular/cellular changes in its structure, providing conditional access to the genetic machinery. ATP-dependent chromatin modifiers regulate access of transcription factors and RNA polymerases to DNA by either "opening" or "closing" the structure of chromatin, and its aberrant regulation leads to a variety of neurodevelopmental disorders. The chromodomain helicase DNA-binding (CHD) proteins are ATP-dependent chromatin modifiers involved in the organization of chromatin structure, act as gatekeepers of genomic access, and deposit histone variants required for gene regulation. In this review, we first discuss the structural and functional domains of the CHD proteins, and their binding sites, and phosphorylation, acetylation, and methylation sites. The conservation of important amino acids in SWItch/sucrose non-fermenting (SWI/SNF) domains, and their protein and mRNA tissue expression profiles are discussed. Next, we convey the important binding partners of CHD proteins, their protein complexes and activities, and their involvements in epigenetic regulation. We also show the ChIP-seq binding dynamics for CHD1, CHD2, CHD4, and CHD7 proteins at promoter regions of histone genes, as well as several genes that are critical for neurodevelopment. The role of CHD proteins in development is also discussed. Finally, this review provides information about CHD protein mutations reported in autism and neurodevelopmental disorders, and their pathogenicity. Overall, this review provides information on the progress of research into CHD proteins, their structural and functional domains, epigenetics, and their role in stem cell, development, and neurological disorders.

Histone H4 variant, H4G, drives ribosomal RNA transcription and breast cancer cell proliferation by loosening nucleolar chromatin structure.

The hominidae-specific histone variant H4G is expressed in breast cancer patients in a stage-dependent manner. H4G localizes primarily in the nucleoli via its interaction with nucleophosmin (NPM1). H4G is involved in rDNA transcription and ribosome biogenesis, which facilitates breast cancer cell proliferation. However, the molecular mechanism underlying this process remains unknown. Here, we show that H4G is not stably incorporated into nucleolar chromatin, even with the chaperoning assistance of NPM1. H4G likely form transient nucleosome-like-structure that undergoes rapid dissociation. In addition, the nucleolar chromatin in H4GKO cells is more compact than WT cells. Altogether, our results suggest that H4G relaxes the nucleolar chromatin and enhances rRNA transcription by forming destabilized nucleosome in breast cancer cells.

Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells.

We observed prominent effects of doxorubicin (Dox), an anthracycline widely used in anti-cancer therapy, on the aggregation and intracellular distribution of both partners of the H2A-H2B dimer, with marked differences between the two histones. Histone aggregation, assessed by Laser Scanning Cytometry via the retention of the aggregates in isolated nuclei, was observed in the case of H2A. The dominant effect of the anthracycline on H2B was its massive accumulation in the cytoplasm of the Jurkat leukemia cells concomitant with its disappearance from the nuclei, detected by confocal microscopy and mass spectrometry. A similar effect of the anthracycline was observed in primary human lymphoid cells, and also in monocyte-derived dendritic cells that harbor an unusually high amount of H2B in their cytoplasm even in the absence of Dox treatment. The nucleo-cytoplasmic translocation of H2B was not affected by inhibitors of major biochemical pathways or the nuclear export inhibitor leptomycin B, but it was completely diminished by PYR-41, an inhibitor with pleiotropic effects on protein degradation pathways. Dox and PYR-41 acted synergistically according to isobologram analyses of cytotoxicity. These large-scale effects were detected already at Dox concentrations that may be reached in the typical clinical settings, therefore they can contribute both to the anti-cancer mechanism and to the side-effects of this anthracycline.

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2.

BACKGROUND: MeCP2-a chromatin-binding protein associated with Rett syndrome-has two main isoforms, MeCP2-E1 and MeCP2-E2, differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest that they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. RESULTS: Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl-binding domain (MBD) to interact with DNA as well as influencing the turn-over rates, binding dynamics, response to neuronal depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. CONCLUSIONS: Our study supports the idea that Rett syndrome might arise from simultaneous impairment of cellular processes involving non-overlapping functions of MECP2 isoforms. For instance, MeCP2-E1 mutations might impact stimuli-dependent chromatin regulation, while MeCP2-E2 mutations could result in aberrant ribosomal expression. Overall, our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.

A novel histone H4 variant H4G regulates rDNA transcription in breast cancer.

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. We found that H4G is expressed in a variety of human cell lines and exhibit tumor-stage dependent overexpression in tissues from breast cancer patients. We found that H4G localized primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha-helix 3 of the histone fold motif with a histone chaperone, nucleophosmin 1. In addition, we found that modulating H4G expression affects rRNA expression levels, protein synthesis rates and cell-cycle progression. Our data suggest that H4G expression alters nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

Alterations in the properties of sperm protamine-like II protein after exposure of Mytilus galloprovincialis (Lamarck 1819) to sub-toxic doses of cadmium.

Protamine-like proteins (PL-II, PL-III and PL-IV) represent the major basic nuclear component of Mytilus galloprovincialis L sperm chromatin. The present study investigates the effects induced on the properties of PL-II protein after exposure of Mytilus galloprovincialis L for 24 h to 1.5 and 5 µM CdCl2. We found cadmium accumulation in protamine-like proteins with a linear grow up with the exposition dose. In particular, after 5 µM CdCl2 mussels exposure, the mobility of PL-II band changed in SDS-PAGE, suggesting structural rearrangement in presence of cadmium. Structural analysis using fluorescent probes, indicated that at 5 µM CdCl2 the complete conformational change of PL-II protein was reached. In the same condition of mussels exposure of 5 µM CdCl2, PL-II protein changed its DNA binding mode, which determined a closer DNA binding, because higher amount of NaCl were required for PL-II protein release by sperm nuclei. These results supported the hypothesis that mussel exposure to this CdCl2 dose, although lower to toxic ones, affects the properties of this protein and as a consequence chromatin organization of spermatozoa that is essential for the success of fertilization.

Protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on male mollusk Mytilus galloprovincialis (Lamarck 1819).

Here we report the industrial pollution effects due to cadmium on the reproductive health of Mytilus galloprovincialis. Mussels were removed from the biofouling of a Conatex panel after one year exposition at a polluted site near a disposal metallurgical factory. A high cadmium bioaccumulation was observed in the testis of mussels housed at the polluted site, with respect to a control site, as determined by inductively coupled plasma-mass spectrometry, along with a 10 fold increase in metallothionein 20 kDa gene (mt20) expression levels determined by qPCR. Furthermore, mussels transferred into laboratory tanks from the reference site, and exposed to 1.5, 5 and 10 µM CdCl2, revealed a 1.7, 3.2 and 4.5 fold expression increase in the testis mt20, respectively, and a positive correlation with cadmium bioaccumulation was found. To evaluate a potential detrimental risk of such alterations on spermatozoa, we carried out electrophoretic analyses on their protamine-like proteins. As determined by AU-PAGE, after 1.5 µM CdCl2 exposure, protamine-like proteins also display major alterations with respect to those obtained after 5 and 10 µM CdCl2 exposure. All protamine-like proteins isolated from the polluted biofouling were in an aggregated form and displayed the same reduced DNA binding affinity of the protamine-like proteins obtained after 1.5 µM CdCl2 as demonstrated EMSA with sperm genomic DNA. Our results contribute to the studies concerning cadmium induced testis alterations and highlight protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on Mytilus galloprovincialis, for the sensitivity of the in vivo and in vitro changes of protamine-like proteins' state and their DNA binding affinity.

Metformin alters H2A.Z dynamics and regulates androgen dependent prostate cancer progression.

Epigenetic mechanisms involved in prostate cancer include hypermethylation of tumor suppressor genes, general hypomethylation of the genome, and alterations in histone posttranslational modifications (PTMs). In addition, over expression of the histone variant H2A.Z as well as deregulated expression of Polycomb group proteins including EZH2 have been well-documented. Recent evidence supports a role for metformin in prostate cancer (PCa) treatment. However, the mechanism of action of metformin in PCa is poorly understood. We provide data showing that metformin epigenetically targets PCa by altering the levels and gene binding dynamics of histone variant H2A.Z. Moreover, we show that the increase in H2A.Z upon metformin treatment occurs preferentially due to H2A.Z.1 isoform. Chromatin immunoprecipitation (ChIP)-RT PCR analysis indicates that metformin treatment results in an increased H2A.Z occupancy on the androgen receptor (AR) and AR-regulated genes that is more prominent in the androgen dependent AR positive LNCaP cells. Repression of H2A.Z.1 gene by siRNA-mediated knock down identified this H2A.Z isoform to be responsible. Based on preliminary data with an EZH2-specific inhibitor, we suggest that the effects of metformin on the early stages of PCa may involve both EZH2 and H2A.Z through the alteration of different molecular pathways.

Trichostatin A decreases the levels of MeCP2 expression and phosphorylation and increases its chromatin binding affinity.

MeCP2 binds to methylated DNA in a chromatin context and has an important role in cancer and brain development and function. Histone deacetylase (HDAC) inhibitors are currently being used to palliate many cancer and neurological disorders. Yet, the molecular mechanisms involved are not well known for the most part and, in particular, the relationship between histone acetylation and MeCP2 is not well understood. In this paper, we study the effect of the HDAC inhibitor trichostatin A (TSA) on MeCP2, a protein whose dysregulation plays an important role in these diseases. We find that treatment of cells with TSA decreases the phosphorylation state of this protein and appears to result in a higher MeCP2 chromatin binding affinity. Yet, the binding dynamics with which the protein binds to DNA appear not to be significantly affected despite the chromatin reorganization resulting from the high levels of acetylation. HDAC inhibition also results in an overall decrease in MeCP2 levels of different cell lines. Moreover, we show that miR132 increases upon TSA treatment, and is one of the players involved in the observed downregulation of MeCP2.

Nucleosome stability measured in situ by automated quantitative imaging.

Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

Barcelona Conference on Epigenetics and Cancer: 50 years of histone acetylation.

The Barcelona Conference on Epigenetics and Cancer (BCEC) was held in Barcelona, Spain, on October 1(st) and 2(nd), 2014. The meeting was co-organized by the Cancer Epigenetics and Biology Program (PEBC-IDIBELL) and B·Debate, an initiative of Biocat, with the support of "la Caixa" Foundation. The scientific committee was comprised of leading scientists in the field of epigenetics: Dr. Manel Esteller, director of PEBC-IDIBELL, Dr. Alejandro Vaquero and Dr. Esteban Ballestar, from PEBC-IDIBELL, Juan Ausió from the University of Victoria (Canada), and Marcus Buschbeck, from the Institute of Predictive and Personalized Medicine of Cancer (IMPPC), as BCEC series coordinator. This meeting was the second edition of the BCEC series, which was launched by 5 leading Barcelonan institutes to bring together leading investigators in the fields of epigenetics and chromatin research. The topics discussed during the meeting included the current challenges, opportunities, and perspectives surrounding the study of histone modifications (focusing in acetylation), chromatin structure and gene expression, and the involvement of histone acetylation in physiology and diseases, such as cancer or neurological diseases.

Comparative structure of vertebrate sperm chromatin.

A consistent feature of sperm nuclei is its exceptionally compact state in comparison with somatic nuclei. Here, we have examined the structural organization of sperm chromatin from representatives of three vertebrate lineages, bony fish (Danio rerio), birds (Gallus gallus domesticus) and mammals (Mus musculus) using light and transmission electron microscopy (TEM). Although the three sperm nuclei are all highly compact, they differ in morphology and in the complement of compaction-inducing proteins. Whereas zebrafish sperm retain somatic histones and a nucleosomal organization, in the rooster and mouse, histones are largely replaced by small, arginine-rich protamines. In contrast to the mouse, the rooster protamine contains no cysteine residues and lacks the potential stabilizing effects of S-S bonds. Protamine driven chromatin compaction results in a stable, highly condensed chromatin, markedly different from the somatic nucleosome-based beads-on-a-string architecture, but its structure remains poorly understood. When prepared gently for whole mount TEM, the rooster and mouse sperm chromatin reveal striking rod-like units 40-50 nm in width. Also present in the mouse, which has very flattened sperm nuclei, but not rooster, where nuclei take the form of elongated cylinders, are toroidal shaped structures, with an external diameter of about 90 nm. In contrast, similarly prepared zebrafish sperm exhibit nucleosomal chromatin. We also examined the early stages in the binding of salmine (the salmon protamine) to defined sequence DNA. These images suggest an initial side-by-side binding of linear DNA-protamine complexes leading to the nucleation of thin, flexible rods with the potential to bend, allowing the ends to come into contact and fuse to form toroidal structures. We discuss the relationship between these in vitro observations and the rods and toroids seen in nuclei, and suggest an explanation for the apparent absence of these structures in TEM images of fully condensed sperm nuclei.

Histone H2A.Z deregulation in prostate cancer. Cause or effect?

Genetic and epigenetic changes are at the root of all cancers. The epigenetic component involves alterations of the post-synthetic modifications of DNA (methylation) and histones (histone posttranslational modifications, PTMs) as well as of those of their molecular "writers," "readers," and "erasers." Noncoding RNAs (ncRNA) can also play a role. Here, we focus on the involvement of histone alterations in cancer, in particular that of the histone variant H2A.Z in the etiology of prostate cancer. The structural mechanisms putatively responsible for the contribution of H2A.Z to oncogenic gene expression programs are first described, followed by what is currently known about the involvement of this histone variant in the regulation of androgen receptor regulated gene expression. The implications of this and their relevance to oncogene deregulation in different stages of prostate cancer, including the progression toward androgen independence, are discussed. This review underscores the increasing awareness of the epigenetic contribution of histone variants to oncogenic progression.

Impaired in vivo binding of MeCP2 to chromatin in the absence of its DNA methyl-binding domain.

MeCP2 is a methyl-CpG-binding protein that is a main component of brain chromatin in vertebrates. In vitro studies have determined that in addition to its specific methyl-CpG-binding domain (MBD) MeCP2 also has several chromatin association domains. However, the specific interactions of MeCP2 with methylated or non-methylated chromatin regions and the structural characteristics of the resulting DNA associations in vivo remain poorly understood. We analysed the role of the MBD in MeCP2-chromatin associations in vivo using an MeCP2 mutant Rett syndrome mouse model (Mecp2(tm1.1Jae)) in which exon 3 deletion results in an N-terminal truncation of the protein, including most of the MBD. Our results show that in mutant mice, the truncated form of MeCP2 (ΔMeCP2) is expressed in different regions of the brain and liver, albeit at 50% of its wild-type (wt) counterpart. In contrast to the punctate nuclear distribution characteristic of wt MeCP2, ΔMeCP2 exhibits both diffuse nuclear localization and a substantial retention in the cytoplasm, suggesting a dysfunction of nuclear transport. In mutant brain tissue, neuronal nuclei are smaller, and ΔMeCP2 chromatin is digested faster by nucleases, producing a characteristic nuclease-resistant dinucleosome. Although a fraction of ΔMeCP2 is found associated with nucleosomes, its interaction with chromatin is transient and weak. Thus, our results unequivocally demonstrate that in vivo the MBD of MeCP2 together with its adjacent region in the N-terminal domain are critical for the proper interaction of the protein with chromatin, which cannot be replaced by any other of its protein domains.

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.

Vertebrate nucleoplasmin and NASP: egg histone storage proteins with multiple chaperone activities.

Recent reviews have focused on the structure and function of histone chaperones involved in different aspects of somatic cell chromatin metabolism. One of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by egg histone storage chaperones. These include members of the nucleoplasmin (NPM2/NPM3), which are preferentially associated with histones H2A-H2B in the egg and the nuclear autoantigenic sperm protein (NASP) families. Interestingly, in addition to binding and providing storage to H3/H4 in the egg and in somatic cells, NASP has been shown to be a unique genuine chaperone for histone H1. This review revolves around the structural and functional roles of these two families of chaperones whose activity is modulated by their own post-translational modifications (PTMs), particularly phosphorylation. Beyond their important role in the remodeling of paternal chromatin in the early stages of embryogenesis, NPM and NASP members can interact with a plethora of proteins in addition to histones in somatic cells and play a critical role in processes of functional cell alteration, such as in cancer. Despite their common presence in the egg, these two histone chaperones appear to be evolutionarily unrelated. In contrast to members of the NPM family, which share a common monophyletic evolutionary origin, the different types of NASP appear to have evolved recurrently within different taxa.

Chromatin specialization in bivalve molluscs: a leap forward for the evaluation of Okadaic Acid genotoxicity in the marine environment.

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.