Histone H3 Variant Research Articles

The localization of centromere protein A is conserved among tissues

Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occurs during meiosis or during development. Here, we first improve the sequence of the ECA11 centromeric region in the EquCab3.0 assembly. Then, to test whether centromere sliding may occur during development, we map the CENP-A binding domains of ECA11 using ChIP-seq in five tissues of different embryonic origin from the four horses of the equine FAANG (Functional Annotation of ANimal Genomes) consortium. Our results demonstrate that the centromere is localized in the same region in all tissues, suggesting that the position of the centromeric domain is maintained during development.

Symmetric inheritance of parental histones contributes to safeguarding the fate of mouse embryonic stem cells during differentiation.

Parental histones, the carriers of posttranslational modifications, are deposited evenly onto the replicating DNA of sister chromatids in a process dependent on the Mcm2 subunit of DNA helicase and the Pole3 subunit of leading-strand DNA polymerase. The biological significance of parental histone propagation remains unclear. Here we show that Mcm2-mutated or Pole3-deleted mouse embryonic stem cells (ESCs) display aberrant histone landscapes and impaired neural differentiation. Mutation of the Mcm2 histone-binding domain causes defects in pre-implantation development and embryonic lethality. ESCs with biased parental histone transfer exhibit increased epigenetic heterogeneity, showing altered histone variant H3.3 and H3K27me3 patterning at genomic sites regulating differentiation genes. Our results indicate that the lagging strand pattern of H3.3 leads to the redistribution of H3K27me3 in Mcm2-2A ESCs. We demonstrate that symmetric parental histone deposition to sister chromatids contributes to cellular differentiation and development.

Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

The histone chaperone function of Daxx is dispensable for embryonic development

Daxx functions as a histone chaperone for the histone H3 variant, H3.3, and is essential for embryonic development. Daxx interacts with Atrx to form a protein complex that deposits H3.3 into heterochromatic regions of the genome, including centromeres, telomeres, and repeat loci. To advance our understanding of histone chaperone activity in vivo, we developed two Daxx mutant alleles in the mouse germline, which abolish the interactions between Daxx and Atrx (DaxxY130A), and Daxx and H3.3 (DaxxS226A). We found that the interaction between Daxx and Atrx is dispensable for viability; mice are born at the expected Mendelian ratio and are fertile. The loss of Daxx-Atrx interaction, however, does cause dysregulated expression of endogenous retroviruses. In contrast, the interaction between Daxx and H3.3, while not required for embryonic development, is essential for postnatal viability. Transcriptome analysis of embryonic tissues demonstrates that this interaction is important for silencing endogenous retroviruses and for maintaining proper immune cell composition. Overall, these results clearly demonstrate that Daxx has both Atrx-dependent and independent functions in vivo, advancing our understanding of this epigenetic regulatory complex.

Centromeric repeats in Citrus sinensis provide new insights into centromeric evolution and the distribution of G-quadruplex structures

Centromeres play a crucial role in ensuring the accurate separation of chromosomes during cell division. Despite the three rounds of genome sequencing technology undergone by Citrus sinensis (sweet orange), the presence of numerous repetitive DNA elements in its genome has led to substantial gaps in centromeric genomic mapping, leaving the composition of centromeric repeats unclear. To address this, we employed a combination of chromatin immunoprecipitation sequencing with the C. sinensis centromere-specific histone H3 variant antibody and centromere-specific bacterial artificial chromosome-3a sequencing to precisely locate the centromeres. This approach allowed us to identify a series of centromere-specific repeats, comprising five tandem repeats and nine long terminal repeat retrotransposons. Through comprehensive bioinformatics analysis, we gained valuable insights into potential centromeric evolution events and discovered the presence of DNA G-quadruplex structures of centromeric repeats in C. sinensis. Altogether, our study not only offers a valuable reference for centromeric genome assembly but also sheds light on the structural characteristics of C. sinensis centromeres.

Centromere-specifying nucleosomes persist in aging mouse oocytes in the absence of nascent assembly

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A.1 In cultured somatic cells, an established paradigm of cell-cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germ line challenges this model because of the cell-cycle arrest between pre-meiotic S phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocytes,2,3 suggesting that a similar process may be required for centromere inheritance in mammals. To test this hypothesis, we developed an oocyte-specific conditional knockout (cKO) mouse for Mis18α, an essential component of the assembly machinery. We find that embryos derived from Mis18α knockout oocytes fail to assemble CENP-A nucleosomes prior to zygotic genome activation (ZGA), validating the knockout model. We show that deletion of Mis18α in the female germ line at the time of birth has no impact on centromeric CENP-A nucleosome abundance, even after 6-8months of aging. In addition, there is no detectable detriment to fertility. Thus, centromere chromatin is maintained long-term, independent of new assembly during the extended prophase I arrest in mouse oocytes.

Histone Variants and Their Chaperones in Hematological Malignancies.

Epigenetic regulation occurs on the level of compacting DNA into chromatin. The functional unit of chromatin is the nucleosome, which consists of DNA wrapped around a core of histone proteins. While canonical histone proteins are incorporated into chromatin through a replication-coupled process, structural variants of histones, commonly named histone variants, are deposited into chromatin in a replication-independent manner. Specific chaperones and chromatin remodelers mediate the locus-specific deposition of histone variants. Although histone variants comprise one of the least understood layers of epigenetic regulation, it has been proposed that they play an essential role in directly regulating gene expression in health and disease. Here, we review the emerging evidence suggesting that histone variants have a role at different stages of hematopoiesis, with a particular focus on the histone variants H2A, H3, and H1. Moreover, we discuss the current knowledge on how the dysregulation of histone variants can contribute to hematopoietic malignancies.

Direct observation of coordinated assembly of individual native centromeric nucleosomes

Eukaryotic chromosome segregation requires the kinetochore, a megadalton‐sized machine that forms on specialized centromeric chromatin containing CENP‐A, a histone H3 variant. CENP‐A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP‐A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP‐A can arrive at centromeres without its dedicated centromere‐specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA‐binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP‐A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.

H3.3B controls aortic dissection progression by regulating vascular smooth muscle cells phenotypic transition and vascular inflammation

Aortic dissection is a devastating cardiovascular disease with a high lethality. Histone variants maintain the genomic integrity and play important roles in development and diseases. However, the role of histone variants in aortic dissection has not been well identified. In the present study, H3f3b knockdown reduced the synthetic genes expression of VSMCs, while overexpressing H3f3b exacerbated the cellular immune response of VSMCs induced by inflammatory cytokines. Combined RNA-seq and ChIP-seq analyses revealed that histone variant H3.3B directly bound to the genes related to extracellular matrix, VSMC synthetic phenotype, cytokine responses and TGFβ signaling pathway, and regulated their expressions. In addition, VSMC-specific H3f3b knockin aggravated aortic dissection development in mice, while H3f3b knockout significantly reduced the incidence of aortic dissection. In term of mechanisms, H3.3B regulated Spp1 and Ccl2 genes, inducing the apoptosis of VSMCs and recruiting macrophages. This study demonstrated the vital roles of H3.3B in phenotypic transition of VSMCs, loss of media VSMCs, and vascular inflammation in aortic dissection.

Misregulation of cell cycle-dependent methylation of budding yeast CENP-A contributes to chromosomal instability.

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.

The linker histone H1-BRCA1 axis is a crucial mediator of replication fork stability.

The maintenance of genome integrity relies on replication fork stabilization upon encountering endogenous and exogenous sources of DNA damage. How this process is coordinated with the local chromatin environment remains poorly defined. Here, we show that the replication-dependent histone H1 variants interact with the tumour suppressor BRCA1 in a replication stress-dependent manner. Transient loss of the replication-dependent histones H1 does not affect fork progression in unchallenged conditions but leads to the accumulation of stalled replication intermediates. Upon challenge with hydroxyurea, cells deficient for histone H1 variants fail to recruit BRCA1 to stalled replication forks and undergo MRE11-dependent fork resection and collapse, which ultimately leads to genomic instability and cell death. In summary, our work defines an essential role of the replication-dependent histone H1 variants in mediating BRCA1-dependent fork protection and genome stability.

DIPG-35. IDENTIFYING DRIVER-SPECIFIC VULNERABILITIES IN PAEDIATRIC HIGH GRADE GLIOMA SUBTYPES

Abstract Paediatric high-grade gliomas (pHGGs) are incurable malignant brain tumours and a leading cause of cancer-related death in children. The majority of pHGGs carry lysine-to-methionine (K27M) or glycine-to-arginine/valine (G34R/V) mutations in histone variants H3.1 or H3.3. Moreover, histone mutations associate with different anatomical locations and co-segregating mutations defining distinct tumour subtypes within pHGG. However whether these co-occuring mutations can act as drivers to modify tumour phenotypes and drug sensitivities is currently unknown. In order to functionally evaluate the role of partner alterations and to identify new therapeutic targets, we developed in vivo tumour models of pHGG subtypes using in utero electroporation (IUE) in combination with piggyBac transposon and CRISPR technology. We also established ex vivo glioma stem cell (GSCs) lines from mouse models with different co-segregating mutations, able to engraft in syngeneic immune-competent mice. Then, we performed transcriptome analysis and drug screening identifying selective pharmacological vulnerabilities. Our approach represents a preclinical platform to evaluate subtype-specific precision therapies identifying new pathways involved in brain tumour initiation, progression and maintenance.

DIPG-21. LINKING DETERMINATION OF CELL FATE IN THE DEVELOPING CENTRAL NERVOUS SYSTEM TO DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. Next-generation sequencing has revealed that the majority of patients with DMG harbor K27M mutations in canonical histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and an oligodendrocyte precursor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required in OPC differentiation in vivo. Co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. The Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, as well as a reduction in migration, invasion, and colony formation in vitro. An RNAseq database containing 45 human DMG cell lines showed an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DLX2high/OLIG1/2low cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2high/DLX2low expressing cell lines underwent stable transfection of an DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone H3 marks (K27me3, K27ac), K27M, and OPC signature genes will be presented along with comparison of altered OPC genes from the Dlx1/Dlx2 DKO mouse as determined by RNAseq. The manipulation of DLX2 expression in human DMG cells both in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

DIPG-48. DIPG THROUGH THE LOOKING GLASS

Abstract Diffuse intrinsic pontine glioma (DIPG) has a peculiar pathogenesis with canonical Histone variants H3.1/H3.2 wherein Lysine 27 (H3K27) is substituted to methionine (H3K27M). It is restricted to pons and affects young children whereas non canonical H3.3 mutations result in tumours across the midline and affects older children. This discrepancy in the spatiotemporal distribution provides cues to target DIPG. Based on altered neuronal signalling which is a hallmark of cancer, a deeper understanding of neuron tumour interaction may unravel numerous new targets. One such target is light. The restriction of specific spectrum of light may hinder the progression and even the development of tumours in young children. It provides a lucrative therapeutic option which can be implemented merely by blocking a certain spectrum of light with glasses in an otherwise baffling disease. It warrants a deeper understanding of the light exposure on DIPG tumour progression.

IMMU-04. TARGETING ALTERNATIVE SPLICING VARIANTS TO STIMULATE ANTI-TUMOR IMMUNITY IN A NOVEL SYNGENEIC MOUSE MODEL OF DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT

Abstract Juvenile presentation of HGG has been shown to frequently involve somatic mutations of the H3-3A gene, which encodes a histone H3 variant. To address the paucity of identified TSAs in DHG, we utilized a novel computational platform, Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS). The IRIS pipeline is designed to leverage large-scale cancer and normal transcriptomic data to identify peptide sequences that result from alternative splicing events that are highly enriched in cancer cells, and are predicted to bind the HLA class I alleles carried by the individual patient. To investigate whether peptide-pulsed dendritic cells (DC) together with checkpoint blockade might be a potential treatment modality for diffuse hemispheric glioma, H3 G34-mutant, we have developed a novel mouse model, and utilized a novel bioinformatics pipeline to identify tumor-associated peptide antigens. We used the RCAS/tv-A system to target the expression of H3/G34R and PDGF-B and to knock out p53 in neural progenitors in transgenic C57BL/6 neonatal mice. Three independent cell lines were obtained that expressed transcripts associated with oligodendrocyte and interneuron lineages, and formed lethal tumors after intracranial implantation. Implementing IRIS, we identified nonapeptides generated from alternative splicing of mRNAs that were highly enriched in the tumor transcriptomes of two H3/G34R patients, and predicted to bind the HLA-A*2:01 class I allele carried by these patients. Three of the candidate peptides were conserved in the mouse and also predicted to bind the H2-Kb MHC I allele carried by C57/BL6 mice. Using our syngeneic mouse model, we showed that DCs pulsed with these peptides together with anti-PD1 mAb provided a significant survival benefit compared with checkpoint inhibitor and DCs pulsed with peptides that were not predicted to bind the H2-Kb allele.

Reduced histone gene copy number disrupts Drosophila Polycomb function.

The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.

Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores

Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae, we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5’−3’exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.

A rapidly evolving single copy histone H1 variant is associated with male fertility in a parasitoid wasp.

The linker histone H1 binds to the nucleosome core particle at the site where DNA enters and exits, and facilitates folding of the nucleosomes into a higher-order chromatin structure in eukaryotes. Additionally, some variant H1s promote specialized chromatin functions in cellular processes. Germline-specific H1 variants have been reported in some model species with diverse roles in chromatin structure changes during gametogenesis. In insects, the current understanding of germline-specific H1 variants comes mainly from the studies in Drosophila melanogaster, and the information on this set of genes in other non-model insects remains largely unknown. Here, we identify two H1 variants (PpH1V1 and PpH1V2) that are specifically predominantly expressed in the testis of the parasitoid wasp Pteromalus puparum. Evolutionary analyses suggest that these H1 variant genes evolve rapidly, and are generally maintained as a single copy in Hymenoptera. Disruption of PpH1V1 function in the late larval stage male by RNA interference experiments has no phenotype on spermatogenesis in the pupal testis, but results in abnormal chromatin structure and low sperm fertility in the adult seminal vesicle. In addition, PpH1V2 knockdown has no detectable effect on spermatogenesis or male fertility. Collectively, our discovery indicates distinct functions of male germline-enriched H1 variants between parasitoid wasp Pteromalus and Drosophila, providing new insights into the role of insect H1 variants in gametogenesis. This study also highlights the functional complexity of germline-specific H1s in animals.

EWSR1 maintains centromere identity

The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif invitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.