Histone H3 Variant Research Articles

Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres.

Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A–H4–HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48–Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A–H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.

CENP-A Is Dispensable for Mitotic Centromere Function after Initial Centromere/Kinetochore Assembly

Human centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive alphoid DNA sequences. By inducing rapid, complete degradation of endogenous CENP-A, we now demonstrate that once the first steps of centromere assembly have been completed in G1/S, continued CENP-A binding is not required for maintaining kinetochore attachment to centromeres or for centromere function in the next mitosis. Degradation of CENP-A prior to kinetochore assembly is found to block deposition of CENP-C and CENP-N, but not CENP-T, thereby producing defective kinetochores and failure of chromosome segregation. Without the continuing presence of CENP-A, CENP-B binding to alphoid DNA sequences becomes essential to preserve anchoring of CENP-C and the kinetochore to each centromere. Thus, there is a reciprocal interdependency of CENP-A chromatin and the underlyingrepetitive centromere DNA sequences bound by CENP-B in the maintenance of human chromosome segregation.

Characterization of post-translational modifications on lysine 9 of histone H3 variants in mouse testis using matrix-assisted laser desorption/ionization in-source decay.

RationalePost‐translational modifications (PTMs) of histones result in changes to transcriptional activities and chromatin remodeling. Lysine 9 of histone H3 (H3K9) is subject to PTMs, such as methylation and acetylation, which influence histone activity during spermatogenesis. Characterization strategies for studying PTMs on H3K9 have been developed to provide epigenetic and proteomic information. Proteomic analysis has been used to limited success to study PTMs on H3K9; however, a comprehensive analytical approach is required to elucidate global patterns of PTMs of H3 variants during spermatogenesis.MethodsIntact H3 variants in mouse testis were separated by high‐performance liquid chromatography on a reversed‐phase column with an ion‐pairing reagent. Modifications to H3K9 were identified via top‐down analysis using matrix‐assisted laser desorption/ionization in source decay (MALDI‐ISD).ResultsMono‐, di‐, and tri‐methylations were identified at H3K9 in mouse testis and epididymis. These modifications were also observed in testis‐specific histone H3 (H3t). Specifically, tri‐methylation was more abundant on H3tK9 than on K9 of other H3 variants.ConclusionsWe introduce a method for rapid, simple, and comprehensive characterization of PTMs on the N‐termini of H3 variants using MALDI‐ISD. This approach provides novel and useful information, including K9 modifications on H3t, which would benefit epigenetic and proteomic research. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Phenotypic reversion in fas mutants of Arabidopsis thaliana by reintroduction of FAS genes: variable recovery of telomeres with major spatial rearrangements and transcriptional reprogramming of 45S rDNA genes.

Arabidopsis thaliana mutants dysfunctional in the evolutionarily conserved protein complex chromatin assembly factor-1 (CAF-1), which deposits the canonical histone H3 variant H3.1 during DNA synthesis-dependent chromatin assembly, display complex phenotypic changes including meristem and growth alterations, sensitivity to DNA-damaging agents, and reduced fertility. We reported previously that mutants in the FAS1 subunit of CAF-1 progressively lose telomere and 45S rDNA repeats. Here we show that multiple aspects of the fas phenotype are recovered immediately on expression of a reintroduced FAS1 allele, and are clearly independent of the recovery of rDNA copy-numbers and telomeres. In reverted lines, 45S rDNA genes are recovered to diverse levels with a strikingly different representation of their variants, and the typical association of nucleolar organizing region 4 with the nucleolus is perturbed. One of 45S rDNA variants (VAR1), which is silenced in wild-type (WT) plants without mutation history (Col-0 WT), dominates the expression pattern, whereas VAR2 is dominant in Col-0 WT plants. We propose an explanation for the variability of telomere and 45S rDNA repeats associated with CAF-1 function, suggesting that the differences in nuclear partitioning and expression of the rDNA variants in fas mutants and their revertants provide a useful experimental system to study genetic and epigenetic factors in gene dosage compensation.

Dynamic chromatin changes associated with de novo centromere formation in maize euchromatin.

The inheritance and function of centromeres are not strictly dependent on any specific DNA sequence, but involve an epigenetic component in most species. CENH3, a centromere histone H3 variant, is one of the best-described epigenetic factors in centromere identity, but the chromatin features required during centromere formation have not yet been revealed. We previously identified two de novo centromeres on Zea mays (maize) minichromosomes derived from euchromatic sites with high-density gene distributions but low-density transposon distributions. The distribution of gene location and gene expression in these sites indicates that transcriptionally active regions can initiate de novo centromere formation, and CENH3 seeding shows a preference for gene-free regions or regions with no gene expression. The locations of the expressed genes detected were at relatively hypomethylated loci, and the altered gene expression resulted from de novo centromere formation, but not from the additional copy of the minichromosome. The initial overall DNA methylation level of the two de novo regions was at a low level, but increased substantially to that of native centromeres after centromere formation. These results illustrate the dynamic chromatin changes during euchromatin-originated de novo centromere formation, which provides insight into the mechanism of de novo centromere formation and regulation of subsequent consequences.

Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs

SummaryChromatin structure orchestrates the accessibility to the genetic material. Replication-independent histone variants control transcriptional plasticity in postmitotic cells. The life-long accumulation of these histones has been described, yet the implications on organismal aging remain elusive. Here, we study the importance of the histone variant H3.3 in Caenorhabditis elegans longevity pathways. We show that H3.3-deficient nematodes have negligible lifespan differences compared to wild-type animals. However, H3.3 is essential for the lifespan extension of C. elegans mutants in which pronounced transcriptional changes control longevity programs. Notably, H3.3 loss critically affects the expression of a very large number of genes in long-lived nematodes, resulting in transcriptional profiles similar to wild-type animals. We conclude that H3.3 positively contributes to diverse lifespan-extending signaling pathways, with potential implications on age-related processes in multicellular organisms.

Identification of replication-dependent and replication-independent linker histone complexes: Tpr specifically promotes replication-dependent linker histone stability.

BackgroundThere are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. The histone chaperones and other factors that regulate linker histone dynamics in the cell are largely unknown. In particular, it is not known whether replication-dependent and replication-independent linker histones interact with distinct or common sets of proteins. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We then used a variety of in vivo analyses to validate the functional relevance of identified interactions.ResultsWe identified proteins that bind to all linker histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. The nuclear pore complex protein Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability.ConclusionReplication-dependent and replication-independent linker histone variants can interact with both common and distinct sets of proteins. Some of these factors are likely to function as histone chaperones while others may suggest novel links between linker histones and RNA metabolism. The nuclear pore complex protein Tpr specifically interacts with histone H1.1 and H1.2 but not H1x and can regulate the stability of these replication-dependent linker histones.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-016-0074-9) contains supplementary material, which is available to authorized users.

DBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations.

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.

Gene knockdown of CENPA reduces sphere forming ability and stemness of glioblastoma initiating cells

CENPA is a centromere-associated variant of histone H3 implicated in numerous malignancies. However, the role of this protein in glioblastoma (GBM) has not been demonstrated. GBM is one of the most aggressive human cancers. GBM initiating cells (GICs), contained within these tumors are deemed to convey characteristics such as invasiveness and resistance to therapy. Therefore, there is a strong rationale for targeting these cells. We investigated the expression of CENPA and other centromeric proteins (CENPs) in GICs, GBM and variety of other cell types and tissues. Bioinformatics analysis identified the gene signature: high_CENP(AEFNM)/low_CENP(BCTQ) whose expression correlated with significantly worse GBM patient survival.Knockdown of CENPA reduced sphere forming ability, proliferation and cell viability of GICs. We also detected significant reduction in the expression of stemness marker SOX2 and the proliferation marker Ki67. These results indicate that CENPA might represent a promising therapeutic target for GBM treatment.

Insights from biochemical reconstitution into the architecture of human kinetochores.

Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle. Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A). A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN), establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore. Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described, but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.

Modulation of chromatin function through linker histone H1 variants.

In this review, the structural aspects of linker H1 histones are presented as a background for characterization of the factors influencing their function in animal and human chromatin. The action of H1 histone variants is largely determined by dynamic alterations of their intrinsically disordered tail domains, posttranslational modifications and allelic diversification. The interdependent effects of these factors can establish dynamic histone H1 states that may affect the organization and function of chromatin regions.

Nuclear and nucleolar activity of linker histone variant H1.0.

Histone H1.0 belongs to the class of linker histones (H1), although it is substantially distinct from other histone H1 family members. The differences can be observed in the chromosomal location and organization of the histone H1.0 encoding gene, as well as in the length and composition of its amino acid chain. Whereas somatic (H1.1–H1.5) histone H1 variants are synthesized in the cell cycle S-phase, histone H1.0 is synthesized throughout the cell cycle. By replacing somatic H1 variants during cell maturation, histone H1.0 is gradually deposited in low dividing cells and achieves the highest level of expression in the terminally differentiated cells. Compared to other differentiation-specific H1 histone (H5) characteristic for unique tissue and organisms, the distribution of histone H1.0 remains non-specific. Classic investigations emphasize that histone H1.0 is engaged in the organization of nuclear chromatin accounting for formation and maintenance of its nucleosomal and higher-order structure, and thus influences gene expression. However, the recent data confirmed histone H1.0 peculiar localization in the nucleolus and unexpectedly revealed its potential for regulation of nucleolar, RNA-dependent, activity via interaction with other proteins. According to such findings, histone H1.0 participates in the formation of gene-coded information through its control at both transcriptional and translational levels. In order to reappraise the biological significance of histone H1.0, both aspects of its activity are presented in this review.

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 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 Holliday Junction Recognition Protein (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.

HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3.

Chromatin remodelling is essential for cardiac development. Interestingly, the role of histone chaperones has not been investigated in this regard. HIRA is a member of the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex that deposits the variant histone H3.3 on chromatin independently of replication. Lack of HIRA has general effects on chromatin and gene expression dynamics in embryonic stem cells and mouse oocytes. Here we describe the conditional ablation of Hira in the cardiogenic mesoderm of mice. We observed surface oedema, ventricular and atrial septal defects and embryonic lethality. We identified dysregulation of a subset of cardiac genes, notably upregulation of troponins Tnni2 and Tnnt3, involved in cardiac contractility and decreased expression of Epha3, a gene necessary for the fusion of the muscular ventricular septum and the atrioventricular cushions. We found that HIRA binds GAGA rich DNA loci in the embryonic heart, and in particular a previously described enhancer of Tnni2/Tnnt3 (TTe) bound by the transcription factor NKX2.5. HIRA-dependent H3.3 enrichment was observed at the TTe in embryonic stem cells (ESC) differentiated toward cardiomyocytes in vitro. Thus, we show here that HIRA has locus-specific effects on gene expression and that histone chaperone activity is vital for normal heart development, impinging on pathways regulated by an established cardiac transcription factor.

The NnCenH3 protein and centromeric DNA sequence profiles of Nelumbo nucifera Gaertn. (sacred lotus) reveal the DNA structures and dynamics of centromeres in basal eudicots

Centromeres on eukaryotic chromosomes consist of large arrays of DNA repeats that undergo very rapid evolution. Nelumbo nucifera Gaertn. (sacred lotus) is a phylogenetic relict and an aquatic perennial basal eudicot. Studies concerning the centromeres of this basal eudicot species could provide ancient evolutionary perspectives. In this study, we characterized the centromeric marker protein NnCenH3 (sacred lotus centromere-specific histone H3 variant), and used a chromatin immunoprecipitation (ChIP)-based technique to recover the NnCenH3 nucleosome-associated sequences of sacred lotus. The properties of the centromere-binding protein and DNA sequences revealed notable divergence between sacred lotus and other flowering plants, including the following factors: (i) an NnCenH3 alternative splicing variant comprising only a partial centromere-targeting domain, (ii) active genes with low transcription levels in the NnCenH3 nucleosomal regions, and (iii) the prevalence of the Ty1/copia class of long terminal repeat (LTR) retrotransposons in the centromeres of sacred lotus chromosomes. In addition, the dynamic natures of the centromeric region showed that some of the centromeric repeat DNA sequences originated from telomeric repeats, and a pair of centromeres on the dicentric chromosome1 was inactive in the metaphase cells of sacred lotus. Our characterization of the properties of centromeric DNA structure within the sacred lotus genome describes a centromeric profile in ancient basal eudicots and might provide evidence of the origins and evolution of centromeres. Furthermore, the identification of centromeric DNA sequences is of great significance for the assembly of the sacred lotus genome.

H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum

Histones, by packaging and organizing the DNA into chromatin, serve as essential building blocks for eukaryotic life. The basic structure of the chromatin is established by four canonical histones (H2A, H2B, H3 and H4), while histone variants are more commonly utilized to alter the properties of specific chromatin domains. H3.3, a variant of histone H3, was found to have diverse localization patterns and functions across species but has been rather poorly studied in protists. Here we present the first genome-wide analysis of H3.3 in the malaria-causing, apicomplexan parasite, P. falciparum, which revealed a complex occupancy profile consisting of conserved and parasite-specific features. In contrast to other histone variants, PfH3.3 primarily demarcates euchromatic coding and subtelomeric repetitive sequences. Stable occupancy of PfH3.3 in these regions is largely uncoupled from the transcriptional activity and appears to be primarily dependent on the GC-content of the underlying DNA. Importantly, PfH3.3 specifically marks the promoter region of an active and poised, but not inactive antigenic variation (var) gene, thereby potentially contributing to immune evasion. Collectively, our data suggest that PfH3.3, together with other histone variants, indexes the P. falciparum genome to functionally distinct domains and contribute to a key survival strategy of this deadly pathogen.

Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch.

Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

Chromatin assembly: Journey to the CENter of the chromosome.

All eukaryotic genomes are packaged into basic units of DNA wrapped around histone proteins called nucleosomes. The ability of histones to specify a variety of epigenetic states at defined chromatin domains is essential for cell survival. The most distinctive type of chromatin is found at centromeres, which are marked by the centromere-specific histone H3 variant CENP-A. Many of the factors that regulate CENP-A chromatin have been identified; however, our understanding of the mechanisms of centromeric nucleosome assembly, maintenance, and reorganization remains limited. This review discusses recent insights into these processes and draws parallels between centromeric and noncentromeric chromatin assembly mechanisms.

The inheritance of centromere identity

ABSTRACTCENP-A (Centromere protein A) is a histone H3 variant that epigenetically determines the centromere position, but the mechanism of its centromere inheritance is obscure. We propose that CENP-A ubiquitylation, which is inherited through dimerization between rounds of cell division, is a candidate for the epigenetic mark of centromere identity.

Nucleosome disassembly during human non-homologous end joining followed by concerted HIRA- and CAF-1-dependent reassembly.

The cell achieves DNA double-strand break (DSB) repair in the context of chromatin structure. However, the mechanisms used to expose DSBs to the repair machinery and to restore the chromatin organization after repair remain elusive. Here we show that induction of a DSB in human cells causes local nucleosome disassembly, apparently independently from DNA end resection. This efficient removal of histone H3 from the genome during non-homologous end joining was promoted by both ATM and the ATP-dependent nucleosome remodeler INO80. Chromatin reassembly during DSB repair was dependent on the HIRA histone chaperone that is specific to the replication-independent histone variant H3.3 and on CAF-1 that is specific to the replication-dependent canonical histones H3.1/H3.2. Our data suggest that the epigenetic information is re-established after DSB repair by the concerted and interdependent action of replication-independent and replication-dependent chromatin assembly pathways.