Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory.

Histone proteins affect gene expression through multiple mechanisms, including through exchange with histone variants. Recent findings link histone variants to neurological disorders, yet few are well studied in the brain. Most notably, widely expressed variants of H2B remain elusive. We applied recently developed antibodies, biochemical assays, and sequencing approaches to reveal broad expression of the H2B variant H2BE and defined its role in regulating chromatin structure, neuronal transcription, and mouse behavior. We find that H2BE is enriched at promoters, and a single unique amino acid allows it to dramatically enhance chromatin accessibility. Further, we show that H2BE is critical for synaptic gene expression and long-term memory. Together, these data reveal a mechanism linking histone variants to chromatin accessibility, transcriptional regulation, neuronal function, and memory. This work further identifies a widely expressed H2B variant and uncovers a single histone amino acid with profound effects on genomic structure.

Studies on Human Cultured Fibroblasts and Cutaneous Squamous Cell Carcinomas Suggest That Overexpression of Histone Variant H2A.J Promotes Radioresistance and Oncogenic Transformation.

Background: Cellular senescence in response to ionizing radiation (IR) limits the replication of damaged cells by causing permanent cell cycle arrest. However, IR can induce pro-survival signaling pathways that reduce the extent of radiation-induced cytotoxicity and promote the development of radioresistance. The differential incorporation of histone variant H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of radiation-induced senescence. However, the precise epigenetic mechanism and function of H2A.J overexpression in response to IR exposure still needs to be elucidated. Methods: Primary (no target, NT) and genetically modified fibroblasts overexpressing H2A.J (H2A.J-OE) were exposed to 20 Gy and analyzed 2 weeks post-IR for radiation-induced senescence by immunohistochemistry and immunofluorescence microscopy. Transcriptome signatures were analyzed in (non-)irradiated NT and H2A.J-OE fibroblasts by RNA sequencing. Since H2A.J plays an important role in the epidermal homeostasis of human skin, the oncogenic potential of H2A.J was investigated in cutaneous squamous cell carcinoma (cSCC). The tissue microarrays of cSCC were analyzed for H2A.J protein expression pattern by automated image analysis. Results: In response to radiation-induced DNA damage, the overexpression of H2A.J impairs the formation of senescence-associated heterochromatin foci (SAHF), thereby inhibiting the SAHF-mediated silencing of proliferation-promoting genes. The dysregulated activation of cyclins and cyclin-dependent kinases disturbs cell cycle arrest in irradiated H2A.J-OE fibroblasts, thereby overcoming radiation-induced senescence. Comparative transcriptome analysis revealed significantly increased WNT16 signaling in H2A.J OE fibroblasts after IR exposure, promoting the fundamental mechanisms of tumor development and progression, including the activation of the epithelial-mesenchymal transition. The quantitative analysis of cSCCs revealed that undifferentiated tumors are associated with high nuclear H2A.J expression, related with greater oncogenic potential. Conclusion: H2A.J overexpression induces radioresistance and promotes oncogenic transformation through the activation of WNT16 signaling pathway functions. H2A.J-associated signatures may improve risk stratification by identifying patients with more aggressive cSCC who may require radiotherapy with increased doses.

Structural and Biochemical Characterization of the Nucleosome Containing Variants H3.3 and H2A.Z.

Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 influences chromatin dynamics at transcription start sites, and its role in gene regulation, remain elusive. Using a combination of biochemistry and cryo-electron microscopy (cryo-EM), we show that the inclusion of H3.3 alone does not induce discernible changes in nucleosome DNA dynamics. Conversely, the presence of both H3.3 and H2A.Z enhances DNA's flexibility similarly to H2A.Z alone. Interestingly, our findings suggest that the presence of H3.3 in the H2A.Z nucleosome provides slightly increased protection to DNA at internal sites within the nucleosome. These results imply that while H2A.Z at active promoters promotes the formation of more accessible nucleosomes with increased DNA accessibility to facilitate transcription, the simultaneous presence of H3.3 offers an additional mechanism to fine-tune nucleosome accessibility and the chromatin environment.

Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner, independent from MSL complex spreading.

Dosage compensation in Drosophila involves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions of MSL3 (male-specific lethal 3) with histone marks; in particular, Set2-dependent H3 lysine-36 trimethylation (H3K36me3). However, Set2 might affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Further, it is difficult to parse male-specific effects from those that are simply X-specific. To discriminate among these possibilities, we employed genomic approaches in H3K36 (residue) and Set2 (writer) mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global male-specific reduction of X-genes in Set2/H3K36 mutants, the effects were heterogeneous. We identified cohorts of genes whose expression was significantly altered following loss of H3K36 or Set2, but the changes were in opposite directions, suggesting that H3K36me states have reciprocal functions. In contrast to H4K16R controls, analysis of combined H3.2K36R/H3.3K36R mutants neither showed consistent reduction in X-gene expression, nor any correlation with MSL3 binding. Examination of other developmental stages/tissues revealed additional layers of context-dependence. Our studies implicate BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 directly recruits the MSL complex. We propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.

Interplay between histone variants and chaperones in plants.

Histone chaperones and histone variants play crucial roles in DNA replication, gene transcription, and DNA repair in eukaryotes. Histone chaperones reversibly promote nucleosome assembly and disassembly by incorporating or evicting histones and histone variants to modulate chromatin accessibility, thereby altering the chromatin states and modulating DNA-related biological processes. Cofactors assist histone chaperones to target specific chromatin regions to regulate the exchange of histones and histone variants. In this review, we summarize recent progress in the interplay between histone variants and chaperones in plants. We discuss the structural basis of chaperone-histone complexes and the mechanisms of their cooperation in regulating gene transcription and plant development.

The roles and mechanisms of histone variant H2A.Z in transcriptional regulation.

H2A.Z, one of the most well-known variants of histone H2A, has been extensively investigated on its dual roles in gene transcription in recent years. In this review, we focus on the intricate involvement of H2A.Z in transcriptional regulation, including the assembly of distinct H2A.Z subtypes, post-translational modifications and genomic distributions. Emphasis is placed on the biological and pathophysiological implications, particularly in tumorigenesis and nervous system development. We summarize the dynamic regulatory mechanisms governing H2A.Z deposition or eviction on chromatin to provide insights for understanding the diversity of histone variants and promoting the search of new targets in concerned disease diagnosis and treatment.

Incorporation of the histone variant H2A.Z counteracts gene silencing mediated by H3K27 trimethylation in Fusarium fujikuroi.

BACKGROUND: Fusarium fujikuroi is a pathogen of rice causing diverse disease symptoms such as 'bakanae' or stunting, most likely due to the production of various natural products (NPs) during infection. Fusaria have the genetic potential to synthesize a plethora of these compounds with often diverse bioactivity. The capability to synthesize NPs exceeds the number of those being produced by far, implying a gene regulatory network decisive to induce production. One such regulatory layer is the chromatin structure and chromatin-based modifications associated with it. One prominent example is the exchange of histones against histone variants such as the H2A variant H2A.Z. Though H2A.Z already is well studied in several model organisms, its regulatory functions are not well understood. Here, we used F. fujikuroi as a model to explore the role of the prominent histone variant FfH2A.Z in gene expression within euchromatin and facultative heterochromatin. RESULTS: Through the combination of diverse '-omics' methods, we show the global distribution of FfH2A.Z and analyze putative crosstalks between the histone variant and two prominent histone marks, i.e., H3K4me3 and H3K27me3, important for active gene transcription and silencing, respectively. We demonstrate that, if FfH2A.Z is positioned at the + 1-nucleosome, it poises chromatin for gene transcription, also within facultative heterochromatin. Lastly, functional characterization of FfH2A.Z overexpression and depletion mutants revealed that FfH2A.Z is important for wild type-like fungal development and secondary metabolism. CONCLUSION: In this study, we show that the histone variant FfH2A.Z is a mark of positive gene transcription and acts independently of the chromatin state most likely through the stabilization of the + 1-nucleosome. Furthermore, we demonstrate that FfH2A.Z depletion does not influence the establishment of both H3K27me3 and H3K4me3, thus indicating no crosstalk between FfH2A.Z and both histone marks. These results highlight the manifold functions of the histone variant FfH2A.Z in the phytopathogen F. fujikuroi, which are distinct regarding gene transcription and crosstalk with the two prominent histone marks H3K27me3 and H3K4me3, as proposed for other model organisms.

Chromatin sensing: integration of environmental signals to reprogram plant development through chromatin regulators.

Chromatin regulation in eukaryotes plays pivotal roles in controlling the developmental regulatory gene network. This review explores the intricate interplay between chromatin regulators and environmental signals, elucidating their roles in shaping plant development. As sessile organisms, plants have evolved sophisticated mechanisms to perceive and respond to environmental cues, orchestrating developmental programs that ensure adaptability and survival. A central aspect of this dynamic response lies in the modulation of versatile gene regulatory networks, mediated in part by various chromatin regulators. Here, we summarized current understanding of the molecular mechanisms through which chromatin regulators integrate environmental signals, influencing key aspects of plant development.

The Histone Variant H3.3 Is Required for Plant Growth and Fertility in Arabidopsis.

Histones are the core components of the eukaryote chromosome, and have been implicated in transcriptional gene regulation. There are three major isoforms of histone H3 in Arabidopsis. Studies have shown that the H3.3 variant is pivotal in modulating nucleosome structure and gene transcription. However, the function of H3.3 during development remains to be further investigated in plants. In this study, we disrupted all three H3.3 genes in Arabidopsis. Two triple mutants, h3.3cr-4 and h3.3cr-5, were created by the CRISPR/Cas9 system. The mutant plants displayed smaller rosettes and decreased fertility. The stunted growth of h3.3cr-4 may result from reduced expression of cell cycle regulators. The shorter stamen filaments, but not the fertile ability of the gametophytes, resulted in reduced fertility of h3.3cr-4. The transcriptome analysis suggested that the reduced filament elongation of h3.3cr-4 was probably caused by the ectopic expression of several JASMONATE-ZIM DOMAIN (JAZ) genes, which are the key repressors of the signaling pathway of the phytohormone jasmonic acid (JA). These observations suggest that the histone variant H3.3 promotes plant growth, including rosette growth and filament elongation.

Lysine-36 of Drosophila histone H3.3 supports adult longevity.

Aging is a multifactorial process that disturbs homeostasis, increases disease susceptibility, and ultimately results in death. Although the definitive set of molecular mechanisms responsible for aging remain to be discovered, epigenetic change over time is proving to be a promising piece of the puzzle. Several post-translational histone modifications have been linked to the maintenance of longevity. Here, we focus on lysine-36 of the replication-independent histone protein, H3.3 (H3.3K36). To interrogate the role of this residue in Drosophila developmental gene regulation, we generated a lysine-to-arginine mutant that blocks the activity of its cognate-modifying enzymes. We found that an H3.3BK36R mutation causes a significant reduction in adult lifespan, accompanied by dysregulation of the genomic and transcriptomic architecture. Transgenic co-expression of wild-type H3.3B completely rescues the longevity defect. Because H3.3 is known to accumulate in nondividing tissues, we carried out transcriptome profiling of young vs aged adult fly heads. The data show that loss of H3.3K36 results in age-dependent misexpression of NF-κB and other innate immune target genes, as well as defects in silencing of heterochromatin. We propose H3.3K36 maintains the postmitotic epigenomic landscape, supporting longevity by regulating both pericentric and telomeric retrotransposons and by suppressing aberrant immune signaling.

Unraveling Histone Loss in Aging and Senescence.

As the global population experiences a notable surge in aging demographics, the need to understand the intricate molecular pathways exacerbated by age-related stresses, including epigenetic dysregulation, becomes a priority. Epigenetic mechanisms play a critical role in driving age-related diseases through altered gene expression, genomic instability, and irregular chromatin remodeling. In this review, we focus on histones, a central component of the epigenome, and consolidate the key findings of histone loss and genome-wide redistribution as fundamental processes contributing to aging and senescence. The review provides insights into novel histone expression profiles, nucleosome occupancy, disruptions in higher-order chromatin architecture, and the emergence of noncanonical histone variants in the aging cellular landscape. Furthermore, we explore the current state of our understanding of the molecular mechanisms of histone deficiency in aging cells. Specific emphasis is placed on highlighting histone degradation pathways in the cell and studies that have explored potential strategies to mitigate histone loss or restore histone levels in aging cells. Finally, in addressing future perspectives, the insights gained from this review hold profound implications for advancing strategies that actively intervene in modulating histone expression profiles in the context of cellular aging and identifying potential therapeutic targets for alleviating a multitude of age-related diseases.

Histone H3.3 variant plays a critical role on zygote-to-oocyst development in malaria parasites.

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes.

Histone Variant H3.3 Controls Arabidopsis Fertility by Regulating Male Gamete Development.

Reprograming of chromatin structures and changes in gene expression are critical for plant male gamete development, and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3-1 is sterile due to male sterility. Transcriptome analysis of young inflorescence has identified 2348 genes downregulated in h3.3-1 mutant, among which 1087 target genes are directly bound by H3.3, especially at their 3' ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation and pollen tube growth. The function of H3.3 in male gamete development is dependent on the Anti-Silencing Factor 1A/1B (ASF1A/1B)-Histone regulator A (HIRA)-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development.

Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies.

BACKGROUND: Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. RESULTS: We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2-which are common events in adult gliomas and myeloid leukemias-also disrupt the formation of PML bodies. CONCLUSIONS: We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

Humanization reveals pervasive incompatibility of yeast and human kinetochore components.

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.

Competitive Chemical Reaction Kinetic Model of Nucleosome Assembly Using the Histone Variant H2A.Z and H2A In Vitro.

Nucleosomes not only serve as the basic building blocks for eukaryotic chromatin but also regulate many biological processes, such as DNA replication, repair, and recombination. To modulate gene expression in vivo, the histone variant H2A.Z can be dynamically incorporated into the nucleosome. However, the assembly dynamics of H2A.Z-containing nucleosomes remain elusive. Here, we demonstrate that our previous chemical kinetic model for nucleosome assembly can be extended to H2A.Z-containing nucleosome assembly processes. The efficiency of H2A.Z-containing nucleosome assembly, like that of canonical nucleosome assembly, was also positively correlated with the total histone octamer concentration, reaction rate constant, and reaction time. We expanded the kinetic model to represent the competitive dynamics of H2A and H2A.Z in nucleosome assembly, thus providing a novel method through which to assess the competitive ability of histones to assemble nucleosomes. Based on this model, we confirmed that histone H2A has a higher competitive ability to assemble nucleosomes in vitro than histone H2A.Z. Our competitive kinetic model and experimental results also confirmed that in vitro H2A.Z-containing nucleosome assembly is governed by chemical kinetic principles.

Genome-wide identification of mammalian cell-cycle invariant and mitotic-specific macroH2A1 domains.

The histone variant macroH2A has been found to play important regulatory roles in genomic processes, especially in regulating transcriptomes. However, whether macroH2A nucleosomes are retained on mitotic chromosomes to enable maintenance of cell-specific transcriptomes is not known. Here, examining mouse embryonic fibroblast cells (NIH-3T3) with native chromatin immunoprecipitation and sequencing (nChIP-seq), we show that the overwhelming majority (~90%) of macroH2A1 domains identified at the G1/S stage are indeed stably retained on mitotic chromosomes. Unexpectedly though, we also find that there are a number of macroH2A domains that are specific for either mitotic or G1/S cells. Notably, more than 7,000 interphase expressed genes flanked by macroH2A1 domains are loaded with macroH2A1 nucleosomes on the mitotic chromosome to form extended domains. Overall, these results reveal that, while the majority of macroH2A1 domains are indeed faithfully transmitted through the mitotic chromosomes, there is a previously unknown cell-cycle dependent exchange of macroH2A1 nucleosomes at numerous genomic loci, indicating the existence of molecular machineries for this dynamically regulated process. We anticipate that these findings will prove to be essential for the integrity of mitotic progression and the maintenance of cellular identity.

Cryo-EM and biochemical analyses of the nucleosome containing the human histone H3 variant H3.8.

Histone H3.8 is a non-allelic human histone H3 variant derived from H3.3. H3.8 reportedly forms an unstable nucleosome, but its structure and biochemical characteristics have not been revealed yet. In the present study, we reconstituted the nucleosome containing H3.8. Consistent with previous results, the H3.8 nucleosome is thermally unstable as compared to the H3.3 nucleosome. The entry/exit DNA regions of the H3.8 nucleosome are more accessible to micrococcal nuclease than those of the H3.3 nucleosome. Nucleosome transcription assays revealed that the RNA polymerase II (RNAPII) pausing around the superhelical location (SHL) -1 position, which is about 60 base pairs from the nucleosomal DNA entry site, is drastically alleviated. On the other hand, the RNAPII pausing around the SHL(-5) position, which is about 20 base pairs from the nucleosomal DNA entry site, is substantially increased. The cryo-electron microscopy structure of the H3.8 nucleosome explains the mechanisms of the enhanced accessibility of the entry/exit DNA regions, reduced thermal stability and altered RNAPII transcription profile.

Recruitment of the Histone Variant MacroH2A1 to the Pericentric Region Occurs upon Chromatin Relaxation and Is Responsible for Major Satellite Transcriptional Regulation.

Heterochromatin formation plays a pivotal role in regulating chromatin organization and influences nuclear architecture and genome stability and expression. Amongst the locations where heterochromatin is found, the pericentric regions have the capability to attract the histone variant macroH2A1. However, the factors and mechanisms behind macroH2A1 incorporation into these regions have not been explored. In this study, we probe different conditions that lead to the recruitment of macroH2A1 to pericentromeric regions and elucidate its underlying functions. Through experiments conducted on murine fibroblastic cells, we determine that partial chromatin relaxation resulting from DNA damage, senescence, or histone hyper-acetylation is necessary for the recruitment of macroH2A1 to pericentric regions. Furthermore, macroH2A1 is required for upregulation of noncoding pericentric RNA expression but not for pericentric chromatin organization. Our findings shed light on the functional rather than structural significance of macroH2A1 incorporation into pericentric chromatin.

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.