Histone Variants and Their Chaperones: An Emerging Epigenetic Mechanism in Neurodevelopment and Neurodevelopmental Disorders.

Neurodevelopment is a highly regulated process that relies on the precise regulation of gene expression. Numerous epigenetic mechanisms contribute and cooperate to ensure the proper execution of developmental gene expression programs. Indeed, disruption of the molecular machinery regulating the deposition or removal of epigenetics markers is associated with numerous neurodevelopmental disorders, including autism spectrum disorder and intellectual disabilities. Among the various epigenetic marks that are fundamental for brain development, research has recently begun to focus on the role of histone variants and their associated chaperone proteins. Replication-independent histone variants can replace replication-dependent canonical histones in neuronal chromatin, giving nucleosomes unique properties that allow them to influence transcription. The deposition and removal of histone variants into neuronal chromatin are controlled by chaperone proteins that are integrated into chromatin remodelling complexes. Several studies report that the deposition and removal of histone variants by chaperone proteins from genes during development is pivotal for the regulation of gene expression, suggesting they are fundamental for neurodevelopment. This review will focus on the histone variants H2A.Z and H3.3, and the exclusive chaperones that regulate their dynamics, in gene expression during neurodevelopment and the progression of neurodevelopmental disorders.

Evolution of a histone variant involved in compartmental regulation of NAD metabolism.

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.

Invasion of phagocytic Galectin 3 expressing macrophages in the diabetic brain disrupts vascular repair.

The cellular events that dictate the repair of damaged vessels in the brain, especially in those with vascular risk factors such as diabetes, is poorly understood. Here, we dissected the role of resident microglia and infiltrative macrophages in determining the repair of ruptured cerebral microvessels. Using in vivo time-lapse imaging, gene expression analysis, and immunohistochemistry, we identified a unique population of phagocytic Galectin 3 (Gal3) expressing macrophages, distinct from resident microglia, which infiltrated and aggregated at the site of injury in diabetic mice and were associated with the elimination of microvessels. Depletion of these infiltrative macrophages in diabetic mice attenuated phagocytic activity and prevented the loss of blood vessels after injury. These findings highlight a previously unknown role for infiltrative Gal3 expressing macrophages in promoting vessel elimination after brain injury and provide impetus for future studies to determine whether depleting these cells can facilitate vascular repair in at risk populations.

Deciphering the Enigma of the Histone H2A.Z-1/H2A.Z-2 Isoforms: Novel Insights and Remaining Questions.

The replication independent (RI) histone H2A.Z is one of the more extensively studied variant members of the core histone H2A family, which consists of many replication dependent (RD) members. The protein has been shown to be indispensable for survival, and involved in multiple roles from DNA damage to chromosome segregation, replication, and transcription. However, its functional involvement in gene expression is controversial. Moreover, the variant in several groups of metazoan organisms consists of two main isoforms (H2A.Z-1 and H2A.Z-2) that differ in a few (3-6) amino acids. They comprise the main topic of this review, starting from the events that led to their identification, what is currently known about them, followed by further experimental, structural, and functional insight into their roles. Despite their structural differences, a direct correlation to their functional variability remains enigmatic. As all of this is being elucidated, it appears that a strong functional involvement of isoform variability may be connected to development.

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.

Protamines from liverwort are produced by post-translational cleavage and C-terminal di-aminopropanelation of several male germ-specific H1 histones.

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.

Spermatogenesis in haploid males of the jewel wasp Nasonia vitripennis.

Males of hymenopteran insects, which include ants, bees and wasps, develop as haploids from unfertilized eggs. In order to accommodate their lack of homologous chromosome pairs, some hymenopterans such as the honeybee have been shown to produce haploid sperm through an abortive meiosis. We employed microscopic approaches to visualize landmark aspects of spermatogenesis in the jewel wasp Nasonia vitripennis, a model for hymenopteran reproduction and development. Our work demonstrates that N. vitripennis, like other examined hymenopterans, exhibits characteristics indicative of an abortive meiosis, including slight enlargement of spermatocytes preceding meiotic initiation. However, we saw no evidence of cytoplasmic buds containing centrioles that are produced from the first abortive meiotic division, which occurs in the honeybee. In contrast to other previously studied hymenopterans, N. vitripennis males produce sperm in bundles that vary widely from 16 to over 200, thus reflecting a range of cellular divisions. Our results highlight interesting variations in spermatogenesis among the hymenopteran insects, and together with previous studies, they suggest a pattern of progression from meiosis to a more mitotic state in producing sperm.

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.

Fetal alcohol spectrum disorder (FASD) affects the hippocampal levels of histone variant H2A.Z-2.

Fetal alcohol spectrum disorder (FASD) is caused by prenatal exposure to ethanol and has been linked to neurodevelopmental impairments. Alcohol has the potential to alter some of the epigenetic components that play a critical role during development. Previous studies have provided evidence that prenatal exposure to ethanol results in abnormal epigenetic patterns (i.e., hypomethylation) of the genome. The aim of this study was to determine how prenatal exposure to ethanol in rats affects the hippocampal levels of expression of two important brain epigenetic transcriptional regulators involved in synaptic plasticity and memory consolidation: methyl CpG-binding protein 2 (MeCP2) and histone variant H2A.Z. Unexpectedly, under the conditions used in this work we were not able to detect any changes in MeCP2. Interestingly, however, we observed a significant decrease in H2A.Z, accompanied by its chromatin redistribution in both female and male FASD rat pups. Moreover, the data from reverse-transcription qPCR later confirmed that this decrease in H2A.Z is mainly due to down-regulation of its H2A.Z-2 isoform gene expression. Altogether, these data provide strong evidence that prenatal exposure to ethanol alters histone variant H2A.Z during neurogenesis of rat hippocampus.

Coral epigenetic responses to nutrient stress: Histone H2A.X phosphorylation dynamics and DNA methylation in the staghorn coral Acropora cervicornis.

Nutrient pollution and thermal stress constitute two of the main drivers of global change in the coastal oceans. While different studies have addressed the physiological effects and ecological consequences of these stressors in corals, the role of acquired modifications in the coral epigenome during acclimatory and adaptive responses remains unknown. The present work aims to address that gap by monitoring two types of epigenetic mechanisms, namely histone modifications and DNA methylation, during a 7-week-long experiment in which staghorn coral fragments (Acropora cervicornis) were exposed to nutrient stress (nitrogen, nitrogen + phosphorus) in the presence of thermal stress. The major conclusion of this experiment can be summarized by two main results: First, coral holobiont responses to the combined effects of nutrient enrichment and thermal stress involve the post-translational phosphorylation of the histone variant H2A.X (involved in responses to DNA damage), as well as nonsignificant modifications in DNA methylation trends. Second, the reduction in H2A.X phosphorylation (and the subsequent potential impairment of DNA repair mechanisms) observed after prolonged coral exposure to nitrogen enrichment and thermal stress is consistent with the symbiont-driven phosphorus limitation previously observed in corals subject to nitrogen enrichment. The alteration of this epigenetic mechanism could help to explain the synergistic effects of nutrient imbalance and thermal stress on coral fitness (i.e., increased bleaching and mortality) while supporting the positive effect of phosphorus addition to improving coral resilience to thermal stress. Overall, this work provides new insights into the role of epigenetic mechanisms during coral responses to global change, discussing future research directions and the potential benefits for improving restoration, management and conservation of coral reef ecosystems worldwide.

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.

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.

A 'selfish' B chromosome induces genome elimination by disrupting the histone code in the jewel wasp Nasonia vitripennis.

Intragenomic conflict describes a phenomenon in which genetic elements act 'selfishly' to gain a transmission advantage at the expense of the whole genome. A non-essential, selfish B chromosome known as Paternal Sex Ratio (PSR) induces complete elimination of the sperm-derived hereditary material in the jewel wasp Nasonia vitripennis. PSR prevents the paternal chromatin from forming chromosomes during the first embryonic mitosis, leading to its loss. Although paternally transmitted, PSR evades self-elimination in order to be inherited. We examined important post-translational modifications to the DNA packaging histones on the normal genome and the PSR chromosome in the fertilized embryo. Three histone marks - H3K9me2,3, H3K27me1, and H4K20me1 - became abnormally enriched and spread to ectopic positions on the sperm's chromatin before entry into mitosis. In contrast, other histone marks and DNA methylation were not affected by PSR, suggesting that its effect on the paternal genome is specific to a subset of histone marks. Contrary to the paternally derived genome, the PSR chromosome was visibly devoid of the H3K27me1 and H4K20me1 marks. These findings strongly suggest that PSR causes paternal genome elimination by disrupting at least three histone marks following fertilization, while PSR avoids self-elimination by evading two of these marks.

Chromatin States in Mouse Sperm Correlate with Embryonic and Adult Regulatory Landscapes.

The mammalian sperm genome is thought to lack substantial information for the regulation of future expression after fertilization. Here, we show that most promoters in mouse sperm are flanked by well-positioned nucleosomes marked by active histone modifications. Analysis of these modifications suggests that many enhancers and super-enhancers functional in embryonic and adult tissues are already specified in sperm. The sperm genome is bound by CTCF and cohesin at sites that are also present in round spermatids and embryonic stem cells (ESCs). These sites mediate interactions that organize the sperm genome into domains and compartments that overlap extensively with those found in mESCs. These results suggest that sperm carry a rich source of regulatory information, encoded in part by its three-dimensional folding specified by CTCF and cohesin. This information may contribute to future expression during embryonic and adult life, suggesting mechanisms by which environmental effects on the paternal germline are transmitted transgenerationally.

Analytical Ultracentrifuge Analysis of Nucleosomes Assembled from Recombinant, Acid-Extracted, HPLC-Purified Histones.

The accumulating discoveries of new posttranslational modifications (PTMs) and the increasing relevance of histone variants within the frame of epigenetics demand the availability of methods for a rapid and efficient nucleosome reconstitution to analyze their structural and functional implications. Here we describe a method suitable for this purpose, starting from bacterially expressed histones, solubilized by acid and purified by reversed-phase high-performance liquid chromatography. This method allows the preparation of micrograms to milligram amounts of in vitro-assembled nucleosomes. Finally, we demonstrate the efficiency of this method for the structural analysis of nucleosomes in the analytical ultracentrifuge.

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 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.

Brain phosphorylation of MeCP2 at serine 164 is developmentally regulated and globally alters its chromatin association.

MeCP2 is a transcriptional regulator whose functional alterations are responsible for several autism spectrum and mental disorders. Post-translational modifications (PTMs), and particularly differential phosphorylation, modulate MeCP2 function in response to diverse stimuli. Understanding the detailed role of MeCP2 phosphorylation is thus instrumental to ascertain how MeCP2 integrates the environmental signals and directs its adaptive transcriptional responses. The evolutionarily conserved serine 164 (S164) was found phosphorylated in rodent brain but its functional role has remained uncharacterized. We show here that phosphorylation of S164 in brain is dynamically regulated during neuronal maturation. S164 phosphorylation highly impairs MeCP2 binding to DNA in vitro and largely affects its nucleosome binding and chromatin affinity in vivo. Strikingly, the chromatin-binding properties of the global MeCP2 appear also extensively altered during the course of brain maturation. Functional assays reveal that proper temporal regulation of S164 phosphorylation controls the ability of MeCP2 to regulate neuronal morphology. Altogether, our results support the hypothesis of a complex PTM-mediated functional regulation of MeCP2 potentially involving a still poorly characterized epigenetic code. Furthermore, they demonstrate the relevance of the Intervening Domain of MeCP2 for binding to DNA.

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.