Histone H3 Variant Research Articles

Histone and DNA methylation control by H3 serine 10/threonine 11 phosphorylation in the mouse zygote

BackgroundIn the mammalian zygote, epigenetic reprogramming is a tightly controlled process of coordinated alterations of histone and DNA modifications. The parental genomes of the zygote show distinct patterns of histone H3 variants and distinct patterns of DNA and histone modifications. The molecular mechanisms linking histone variant-specific modifications and DNA methylation reprogramming during the first cell cycle remain to be clarified.ResultsHere, we show that the degree and distribution of H3K9me2 and of DNA modifications (5mC/5hmC) are influenced by the phosphorylation status of H3S10 and H3T11. The overexpression of the mutated histone variants H3.1 and 3.2 at either serine 10 or threonine 11 causes a decrease in H3K9me2 and 5mC and a concomitant increase in 5hmC in the maternal genome. Bisulphite sequencing results indicate an increase in hemimethylated CpG positions following H3.1T10A overexpression suggesting an impact of H3S10 and H3T11 phosphorylation on DNA methylation maintenance.ConclusionsOur data suggest a crosstalk between the cell-cycle-dependent control of S10 and T11 phosphorylation of histone variants H3.1 and H3.2 and the maintenance of the heterochromatic mark H3K9me2. This histone H3 “phospho-methylation switch” also influences the oxidative control of DNA methylation in the mouse zygote.

EPro-ClearSee: a simple immunohistochemical method that does not require sectioning of plant samples

Investigations into the epigenetic status of individual cells within tissues can produce both epigenetic data for different cell types and positional information of the cells. Thus, these investigations are important for understanding the intra- and inter-cellular control systems of developmental and environmental responses in plants. However, a simple method to detect epigenetic modifications of individual cells in plant tissues is not yet available because detection of the modifications requires immunohistochemistry using specific antibodies. In this study, we developed a simple immunohistochemical method that does not require sectioning to investigate epigenetic modifications. This method uses a clearing system to detect methylated histones, acetylated histones, methylated DNA and/or centromeric histone H3 variants. Analyses of four dicots and five monocots indicated that this method provides a universal technique to investigate epigenetic modifications in diverse plant species.

Erratum to: The role of histone modifications and telomere alterations in the pathogenesis of diffuse gliomas in adults and children.

Genetic profiling is an increasingly useful tool for sub-classification of gliomas in adults and children. Specific gene mutations, structural rearrangements, DNA methylation patterns, and gene expression profiles are now recognized to define molecular subgroups of gliomas that arise in distinct anatomic locations and patient age groups, and also provide a better prediction of clinical outcomes for glioma patients compared to histologic assessment alone. Understanding the role of these distinctive genetic alterations in gliomagenesis is also important for the development of potential targeted therapeutic interventions. Mutations including K27M and G34R/V that affect critical amino acids within the N-terminal tail of the histone H3 variants, H3.3 and H3.1 (encoded by H3F3A and HIST1H3B genes), are prime examples of mutations in diffuse gliomas with characteristic clinical associations that can help diagnostic classification and guide effective patient management. These histone H3 mutations frequently co-occur with inactivating mutations in ATRX in association with alternative lengthening of telomeres. Telomere length can also be maintained through upregulation of telomerase reverse transcriptase (TERT) expression driven by mutation within the TERT gene promoter region, an alteration most commonly found in oligodendrogliomas and primary glioblastomas arising in adults. Interestingly, the genetic alterations perturbing histone and telomere function in pediatric gliomas tend to be different from those present in adult tumors. We present a review of these mutations affecting the histone code and telomere length, highlighting their importance in prognosis and as targets for novel therapeutics in the treatment of diffuse gliomas.

Post-translational modifications of linker histone H1 variants in mammals

The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2–H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

Promiscuous Histone MIS-Assembly is Actively Prevented by Chaperones

Histone proteins are essential for the organization, expression, and inheritance of genetic material for eukaryotic cells. A centromere-specific H3 histone variant, centromere protein A (CENP-A), shares about 50% amino acid sequence identity with H3. CENP-A is required for packaging the centromere and for the proper separation of chromosomes during mitosis. Despite their distinct biological functions, previously reported crystal structures of the CENP-A/H4 and H3/H4 dimers reveal a high degree of similarity. In this work, we characterize the structural dynamics of CENP-A/H4 and H3/H4 dimers based on a dual-resolution approach, using both microsecond-scale explicit-solvent all-atom (AA) and coarse-grained (CG) molecular dynamics (MD) simulations. Our data show that the H4 histone is significantly more rigid compared with the H3 histone and its variant CENP-A, hence, serving as a reinforcing structural element within the histone core. We report that the CENP-A/H4 dimer is significantly more dynamic than its canonical counterpart H3/H4, and our results provide a physical explanation for this flexibility. Further, we observe that the centromere-specific chaperone HJURP stabilizes the CENP-A/H4 dimer by forming a specific electrostatic interaction network. Finally, replacing CENP-A S68 with E68 disrupts the binding interface between CENP-A and HJURP in all-atom MD simulation, and consistently, in vivo experiments demonstrate that replacing CENP-A S68 with E68 disrupts CENP-A's localization to the centromere. Based on all our results, we propose that, during the CENP-A/H4 deposition process, the chaperone HJURP protects various substructures of the dimer, serving both as a folding and binding chaperone.

Defects in methylation of arginine 37 on CENP-A/Cse4 are compensated by the ubiquitin ligase complex Ubr2/Mub1.

The kinetochore, a supramolecular protein complex, provides the physical connection between chromatin and the microtubule and ensures correct chromosome segregation during mitosis. Centromeric regions are marked by the presence of the histone H3 variant CENP-A. Cse4, the CENP-A homologue from Saccharomyces cerevisiae, is methylated on arginine 37 in its N-terminus (R37), and the absence of methylation (cse4-R37A) causes synthetic genetic defects in combination with mutations or deletions in genes encoding components of the Ctf19/CCAN complex and with the CDEI binding protein Cbf1. Here, we report that the absence of the E3 ubiquitin ligase Ubr2 as well as its adaptor protein Mub1 suppresses the defects caused by the absence of Cse4-R37 methylation. Ubr2 is known to regulate the levels of the MIND complex component Dsn1 via ubiquitination and proteasome-mediated degradation. Accordingly, we found that overexpression of DSN1 also led to suppression of Cse4 methylation defects. Altogether, our data indicate that the absence of R37 methylation reduces the recruitment of kinetochore proteins to centromeric chromatin, and that this can be compensated for by stabilising the outer kinetochore protein Dsn1.

The role of histone modifications and telomere alterations in the pathogenesis of diffuse gliomas in adults and children.

Genetic profiling is an increasingly useful tool for sub-classification of gliomas in adults and children. Specific gene mutations, structural rearrangements, DNA methylation patterns, and gene expression profiles are now recognized to define molecular subgroups of gliomas that arise in distinct anatomic locations and patient age groups, and also provide a better prediction of clinical outcomes for glioma patients compared to histologic assessment alone. Understanding the role of these distinctive genetic alterations in gliomagenesis is also important for the development of potential targeted therapeutic interventions. Mutations including K27M and G34R/V that affect critical amino acids within the N-terminal tail of the histone H3 variants, H3.3 and H3.1 (encoded by H3F3A and HIST1H3B genes), are prime examples of mutations in diffuse gliomas with characteristic clinical associations that can help diagnostic classification and guide effective patient management. These histone H3 mutations frequently co-occur with inactivating mutations in ATRX in association with alternative lengthening of telomeres. Telomere length can also be maintained through upregulation of telomerase reverse transcriptase (TERT) expression driven by mutation within the TERT gene promoter region, an alteration most commonly found in oligodendrogliomas and primary glioblastomas arising in adults. Interestingly, the genetic alterations perturbing histone and telomere function in pediatric gliomas tend to be different from those present in adult tumors. We present a review of these mutations affecting the histone code and telomere length, highlighting their importance in prognosis and as targets for novel therapeutics in the treatment of diffuse gliomas.

ATRX and DAXX: Mechanisms and Mutations.

Recent genome sequencing efforts in a variety of cancers have revealed mutations and/or structural alterations in ATRX and DAXX, which together encode a complex that deposits histone variant H3.3 into repetitive heterochromatin. These regions include retrotransposons, pericentric heterochromatin, and telomeres, the latter of which show deregulation in ATRX/DAXX-mutant tumors. Interestingly, ATRX and DAXX mutations are often found in pediatric tumors, suggesting a particular developmental context in which these mutations drive disease. Here we review the functions of ATRX and DAXX in chromatin regulation as well as their potential contributions to tumorigenesis. We place emphasis on the chromatin remodeler ATRX, which is mutated in the developmental disorder for which it is named, α-thalassemia, mental retardation, X-linked syndrome, and at high frequency in a number of adult and pediatric tumors.

CDK-regulated dimerization of M18BP1 on a Mis18 hexamer is necessary for CENP-A loading.

Centromeres are unique chromosomal loci that promote the assembly of kinetochores, macromolecular complexes that bind spindle microtubules during mitosis. In most organisms, centromeres lack defined genetic features. Rather, they are specified epigenetically by a centromere-specific histone H3 variant, CENP-A. The Mis18 complex, comprising the Mis18α:Mis18β subcomplex and M18BP1, is crucial for CENP-A homeostasis. It recruits the CENP-A-specific chaperone HJURP to centromeres and primes it for CENP-A loading. We report here that a specific arrangement of Yippee domains in a human Mis18α:Mis18β 4:2 hexamer binds two copies of M18BP1 through M18BP1's 140 N-terminal residues. Phosphorylation by Cyclin-dependent kinase 1 (CDK1) at two conserved sites in this region destabilizes binding to Mis18α:Mis18β, limiting complex formation to the G1 phase of the cell cycle. Using an improved viral 2A peptide co-expression strategy, we demonstrate that CDK1 controls Mis18 complex recruitment to centromeres by regulating oligomerization of M18BP1 through the Mis18α:Mis18β scaffold.

Histone variant H3.3 and its future prospects in cancer clinic

Histone variant, H3.3 has been a continuous subject of interest in the field of chromatin studies due to its two distinguishing features. First, its incorporation into chromatin is replication-independent, unlike the replication-coupled deposition of its canonical counterparts H3.1/3.2. Second, H3.3 has been consistently associated with an active state of chromatin. Apart from this function research in the past few years has also revealed that H3.3 has a central role to play in maintaining the somatic cell identity, for efficient ultraviolet induce DNA damage repair and proper segregation of chromosomes during cell division. Further, the discovery of “driver mutations” on this variant has bought it to limelight in cancer biology to the extent that “oncohistone,” a new term has been coined for different mutants of H3.3. Here, we review the functional importance of H3.3 in the context of cancer.

Quantitative Microscopy Reveals Centromeric Chromatin Stability, Size, and Cell Cycle Mechanisms to Maintain Centromere Homeostasis.

Centromeres are chromatin domains specified by nucleosomes containing the histone H3 variant, CENP-A. This unique centromeric structure is at the heart of a strong self-templating epigenetic mechanism that renders centromeres heritable. We review how specific quantitative microscopy approaches have contributed to the determination of the copy number, architecture, size, and dynamics of centromeric chromatin and its associated centromere complex and kinetochore. These efforts revealed that the key to long-term centromere maintenance is the slow turnover of CENP-A nucleosomes, a critical size of the chromatin domain and its cell cycle-coupled replication. These features come together to maintain homeostasis of a chromatin locus that directs its own epigenetic inheritance and facilitates the assembly of the mitotic kinetochore.

DNA Sequences in Centromere Formation and Function.

Faithful chromosome segregation during cell division depends on the centromere, a complex DNA/protein structure that links chromosomes to spindle microtubules. This chromosomal domain has to be marked throughout cell division and its chromosomal localization preserved across cell generations. From fission yeast to human, centromeres are established on a series of repetitive DNA sequences and on specialized centromeric chromatin. This chromatin is enriched with the histone H3 variant, named CENP-A, that was demonstrated to be the epigenetic mark that maintains centromere identity and function indefinitely. Although centromere identity is thought to be exclusively epigenetic, the presence of specific DNA sequences in the majority of eukaryotes and of the centromeric protein CENP-B that binds to these sequences, suggests the existence of a genetic component as well. In this review, we will highlight the importance of centromeric sequences for centromere formation and function, and discuss the centromere DNA sequence/CENP-B paradox.

HIRA deficiency in muscle fibers causes hypertrophy and susceptibility to oxidative stress.

Nucleosome assembly proceeds through DNA replication-coupled or replication-independent mechanisms. For skeletal myocytes, whose nuclei have permanently exited the cell cycle, replication-independent assembly is the only mode available for chromatin remodeling. For this reason, any nucleosome composition alterations accompanying transcriptional responses to physiological signals must occur through a DNA replication-independent pathway. HIRA is the histone chaperone primarily responsible for replication-independent incorporation of histone variant H3.3 across gene bodies and regulatory regions. Thus, HIRA would be expected to play an important role in epigenetically regulating myocyte gene expression. The objective of this study was to determine the consequence of eliminating HIRA from mouse skeletal myocytes. At 6 weeks of age, myofibers lacking HIRA showed no pathological abnormalities; however, genes involved in transcriptional regulation were downregulated. By 6 months of age, myofibers lacking HIRA exhibited hypertrophy, sarcolemmal perforation and oxidative damage. Genes involved in muscle growth and development were upregulated, but those associated with responses to cellular stresses were downregulated. These data suggest that elimination of HIRA produces a hypertrophic response in skeletal muscle and leaves myofibers susceptible to stress-induced degeneration.

RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells

The histone chaperone HIRA is involved in depositing histone variant H3.3 into distinct genic regions, including promoters, enhancers, and gene bodies. However, how HIRA deposits H3.3 to these regions remains elusive. Through a short hairpin RNA (shRNA) screening, we identified single-stranded DNA binding protein replication protein A (RPA) as a regulator of the deposition of newly synthesized H3.3 into chromatin. We show that RPA physically interacts with HIRA to form RPA-HIRA-H3.3 complexes, and it co-localizes with HIRA and H3.3 at gene promoters and enhancers. Depletion of RPA1, the largest subunit of the RPA complex, dramatically reduces both HIRA association with chromatin and the deposition of newly synthesized H3.3 at promoters and enhancers and leads to altered transcription at gene promoters. These results support a model whereby RPA, best known for its role in DNA replication and repair, recruits HIRA to promoters and enhancers and regulates deposition of newly synthesized H3.3 to these regulatory elements for gene regulation.

Orchestrating the Specific Assembly of Centromeric Nucleosomes.

Centromeres are chromosomal loci that are defined epigenetically in most eukaryotes by incorporation of a centromere-specific nucleosome in which the canonical histone H3 variant is replaced by Centromere Protein A (CENP-A). Therefore, the assembly and propagation of centromeric nucleosomes are critical for maintaining centromere identify and ensuring genomic stability. Centromeres direct chromosome segregation (during mitosis and meiosis) by recruiting the constitutive centromere-associated network of proteins throughout the cell cycle that in turn recruits the kinetochore during mitosis. Assembly of centromere-specific nucleosomes in humans requires the dedicated CENP-A chaperone HJURP, and the Mis18 complex to couple the deposition of new CENP-A to the site of the pre-existing centromere, which is essential for maintaining centromere identity. Human CENP-A deposition occurs specifically in early G1, into pre-existing chromatin, and several additional chromatin-associated complexes regulate CENP-A nucleosome deposition and stability. Here we review the current knowledge on how new CENP-A nucleosomes are assembled selectively at the existing centromere in different species and how this process is controlled to ensure stable epigenetic inheritance of the centromere.

Centromere Dynamics in Male and Female Germ Cells.

In sexually reproducing organisms the germ line is the cellular lineage that gives rise to gametes. All germ cells originate from germline stem cells that divide asymmetrically to generate gonial pre-cursors, which are amplified in number by mitotic divisions, undergo meiosis and eventually differentiate into mature gametes (haploid eggs and sperm). Information transmitted with gametes is inherited by offspring, and potentially by subsequent generations, instructing in organismal development and beyond. Meiosis comprises one round of DNA replication, followed by two rounds of chromosome segregation; homologous chromosomes segregate in the first division (meiosis I) and sister chromatids segregate in the second division (meiosis II). Important mechanistic features of meiosis occur in substages of prophase I and are critical for genetic recombination, including pairing and synapsis of homologous chromosomes (at leptotene and zygotene), crossing-over (at pachytene), and the appearance of chiasmata (at diplotene/diakinesis). Another unique feature of meiosis is the altered centromere/kinetochore geometry at metaphase I, such that sister kinetochores face the same spindle pole (mono-orientation) and stay together at anaphase I. This chapter reviews centromere dynamics in germ cells, focusing on centromere function and assembly in meiotic cell cycles, as well as centromere inheritance in zygotes. Centromeres are functionally defined by the presence of the histone H3 variant CENP-A, the epigenetic determinant of centromere identity. In most eukaryotes, it is well established that CENP-A function is essential for chromosome segregation in mitosis. CENP-A function in meiosis is less well understood and emerging insights into the differential regulation of meiotic and mitotic CENP-A are discussed.

Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis

Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages ofspermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

CENP-A Modifications on Ser68 and Lys124 Are Dispensable for Establishment, Maintenance, and Long-Term Function of Human Centromeres

CENP-A is a histone H3 variant key to epigenetic specification of mammalian centromeres. Using transient overexpression of CENP-A mutants, two recent reports in Developmental Cell proposed essential centromere functions for post-translational modifications of human CENP-A. Phosphorylation at Ser68 was proposed to have an essential role in CENP-A deposition at centromeres. Blockage of ubiquitination at Lys124 was proposed to abrogate localization of CENP-A to the centromere. Following gene inactivation and replacement in human cells, we demonstrate that CENP-A mutants that cannot be phosphorylated at Ser68 or ubiquitinated at Lys124 assemble efficiently at centromeres during G1, mediate early events in centromere establishment at an ectopic chromosomal locus, and maintain centromere function indefinitely. Thus, neither Ser68 nor Lys124 post-translational modification is essential for long-term centromere identity, propagation, cell-cycle-dependent deposition, maintenance, function, or mediation of early steps in centromere establishment.

Silencing of HJURP induces dysregulation of cell cycle and ROS metabolism in bladder cancer cells via PPARγ-SIRT1 feedback loop.

Holliday Junction Recognition Protein (HJURP) is a centromeric histone chaperone involving in de novo histone H3 variant CenH3 (CENP-A) recruitment. Our transcriptome and in vivo study revealed that HJURP is significantly upregulated in bladder cancer (BCa) tissues at both mRNA and protein levels. Knockdown of HJURP inhibited proliferation and viability of BCa cell lines revealed by CCK-8, colony formation and Ki-67-staining assays, and induced apoptosis and reactive oxygen species (ROS) production, as well as triggered cell cycle arrest at G0/G1 phase possibly via loss of CENP-A. Interestingly, in the HJURP-reduced BCa cells the levels of PPARγ and acetylated-p53 were increased, while the ratio of phosphorylated/total SIRT1 protein was decreased. Moreover, after treatment of the BCa cells using PPARγ antagonist (GW9662) and SIRT1 agonist (resveratrol, RSV) respectively, thee phenotypes of cell cycle arrest, increased ROS production and inhibited proliferation rate were all rescued. Taken together, our results suggested that HJURP might regulate proliferation and apoptosis via the PPARγ-SIRT1 negative feedback loop in BCa cells.

Histone H4 Facilitates the Proteolysis of the Budding Yeast CENP-ACse4 Centromeric Histone Variant.

The incorporation of histone variants into nucleosomes can alter chromatin-based processes. CENP-A is the histone H3 variant found exclusively at centromeres that serves as an epigenetic mark for centromere identity and is required for kinetochore assembly. CENP-A mislocalization to ectopic sites appears to contribute to genomic instability, transcriptional misregulation, and tumorigenesis, so mechanisms exist to ensure its exclusive localization to centromeres. One conserved process is proteolysis, which is mediated by the Psh1 E3 ubiquitin ligase in Saccharomyces cerevisiae (budding yeast). To determine whether there are features of the CENP-A nucleosome that facilitate proteolysis, we performed a genetic screen to identify histone H4 residues that regulate CENP-ACse4 degradation. We found that H4-R36 is a key residue that promotes the interaction between CENP-ACse4 and Psh1 Consistent with this, CENP-ACse4 protein levels are stabilized in H4-R36A mutant cells and CENP-ACse4 is enriched in the euchromatin. We propose that the defects in CENP-ACse4 proteolysis may be related to changes in Psh1 localization, as Psh1 becomes enriched at some 3' intergenic regions in H4-R36A mutant cells. Together, these data reveal a key residue in histone H4 that is important for efficient CENP-ACse4 degradation, likely by facilitating the interaction between Psh1 and CENP-ACse4.