Histone H3 Variant Research Articles

SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin.

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.

The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants.

Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes.

Identification of the Post-translational Modifications Present in Centromeric Chromatin

The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and α-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A. Taken together, our findings provide key information regarding some of the key components of functional centromeric chromatin.

Abstract IA26: Oncogenic mechanisms in diffuse intrinsic pontine gliomas

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with no effective cures. DIPG arises in the brainstem and is found almost exclusively in children. Mutations in canonical cancer signalling pathways including the RTK/RAS/PI3K pathway, the Tp53 pathway, and the CDK4/6 G1 checkpoint occur in DIPGs, targeting different effectors within these pathways and resulting in significant genetic heterogeneity between tumors. However, genome-wide studies provided abundant evidence that unique selective pressures drive HGG in children compared to adults, identifying novel oncogenic mutations connecting tumorigenesis and chromatin regulation as well as developmental signaling pathways. Recurrent mutations of the BMP receptor ACVR1/ALK2 are found in 25-30% of DIPGs, and are further restricted to the youngest patients, highlighting critical connections between development and gliomagenesis. The molecular hallmark of DIPGs is frequent recurrent somatic mutation in histone H3. Mutually exclusive somatic heterozygous H3F3A or HIST1H3B mutations encoding a K27M variant of histone H3.3 or H3.1, respectively, are found in nearly 80 percent of DIPGs. These recurrent mutations provide clear evidence of an important role for chromatin regulation in DIPG disease pathogenesis, and present a unifying molecular mechanism driving DIPG. We have evaluated the contribution of H3.3 K27M mutation to DIPG tumor growth in vitro to gain insights into molecular mechanisms of oncogenic activity, and to assess the potential of histone H3 mutation as a therapeutic sensitivity in this incurable brain tumor. Citation Format: Andre B. Silveira, Lawryn H. Kasper, Jon D. Larson, Xiaoyan Zhu, Alexander K. Diaz, Suzanne J. Baker. Oncogenic mechanisms in diffuse intrinsic pontine gliomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA26.

Compromised Telomeric Heterochromatin Promotes ALTernative Lengthening of Telomeres

Alternative lengthening of telomeres (ALT) is an enigmatic process that allows certain cancers to maintain telomeres in the absence of telomerase. ALT cancers are frequently defective for ATRX/DAXX, a chaperone complex that deposits histone variant H3.3 at telomeres. We propose that mutations in alpha thalassemia-mental retardation syndrome X-linked (ATRX)/death-domain associated protein (DAXX) prime ALT activation by disrupting telomeric heterochromatin.

Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis.

ABSTRACTHIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis.

Structure and Functions of Linker Histones.

Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions- from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.

RbAp46/48(LIN-53) Is Required for Holocentromere Assembly in Caenorhabditis elegans.

Centromeres, the specialized chromosomal regions for recruiting kinetochores and directing chromosome segregation, are epigenetically marked by a centromeric histone H3 variant, CENP-A. To maintain centromere identity through cell cycles, CENP-A diluted during DNA replication is replenished. The licensing factor M18BP1(KNL-2) is known to recruit CENP-A to holocentromeres. Here, we show that RbAp46/48(LIN-53), a conserved histone chaperone, is required for CENP-A(HCP-3) localization in holocentric Caenorhabditis elegans. Indeed, RbAp46/48(LIN-53) and CENP-A(HCP-3) localizations are interdependent. RbAp46/48(LIN-53) localizes to the centromere during metaphase in a CENP-A(HCP-3)- and M18BP1(KNL-2)-dependent manner, suggesting CENP-A(HCP-3) loading may occur before anaphase. RbAp46/48(LIN-53) does not function at the centromere through histone acetylation, H3K27 trimethylation, or its known chromatin-modifying complexes. RbAp46/48(LIN-53) may function independently to escort CENP-A(HCP-3) for holocentromere assembly but is dispensable for other kinetochore protein recruitment. Nonetheless, depletion of RbAp46/48(LIN-53) leads to anaphase bridges and chromosome missegregation. This study unravels the holocentromere assembly hierarchy and its conservation with monocentromeres.

CENP-C directs a structural transition of CENP-A nucleosomes mainly through sliding of DNA gyres.

The histone H3 variant, CENP-A, is incorporated into nucleosomes that mark centromere location. We recently reported that CENP-A confers an altered nucleosome shape relative to its counterparts containing conventional H3. Using a single molecule fluorescence resonance energy transfer (FRET) approach with recombinant human histones and centromere DNA, we now find that the nucleosome shape change that CENP-A directs is dominated by lateral passing of the two DNA gyres (gyre sliding). A non-histone centromere protein, CENP-C, binds to and reshapes the nucleosome, sliding the DNA gyres back to positions similar to those in canonical nucleosomes containing conventional histone H3. The model we generate to explain the CENP-A nucleosome transition provides an example of a shape change imposed by external binding proteins, and has important implications for understanding the epigenetic basis for the faithful inheritance of centromere location on the chromosome.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning.

The consolidation of short-term labile memories for long-term storage requires transcription and there is growing interest in defining the epigenetic mechanisms regulating these transcriptional events. In particular, it has been hypothesized that combinations of histone post-translational modifications (PTMs) have the potential to store memory by dynamically defining the transcriptional status of any given gene loci. Studying epigenetic phenomena during long-term memory consolidation, however, is complicated by the complex cellular heterogeneity of the brain, in which epigenetic signal from memory-relevant cells can be obscured or diluted by the surrounding milieu. To address this issue, we have developed a transgenic mouse line expressing a tetO-regulated, hemagglutinin (HA)-tagged histone H3.3 exclusively in excitatory neurons of the forebrain. Unlike canonical histones, histone H3.3 is incorporated at promoter regions of transcriptionally active genes in a DNA replication-independent manner, stably “barcoding” active regions of the genome in post-mitotic cells. Immunoprecipitating H3.3-HA containing nucleosomes from the hippocampus will therefore enrich for memory-relevant chromatin by isolating actively transcribed regions of the excitatory neuron genome. To evaluate the validity of using H3.3 “barcoding” to sort chromatin, we performed a molecular and behavioral characterization of the H3.3-HA transgenic mouse line. Expectedly, we find that H3.3-HA is incorporated preferentially at promoter regions of actively-transcribed neuronal genes and that expression can be effectively regulated by doxycycline. Additionally, H3.3-HA overexpression does not adversely affect exploratory or anxiety-related behaviors, nor does it affect spatial memory. Transgenic animals do, however, exhibit deficits in contextual memory and motor learning, revealing the importance of this histone isoform in the brain. Future studies in the H3.3-HA transgenic mouse line will define the combinatorial histone PTM landscape during spatial memory consolidation and will investigate the important contributions of histone H3.3 to the normal functioning of the brain.

Inbreeding drives maize centromere evolution

Functional centromeres, the chromosomal sites of spindle attachment during cell division, are marked epigenetically by the centromere-specific histone H3 variant cenH3 and typically contain long stretches of centromere-specific tandem DNA repeats (∼1.8 Mb in maize). In 23 inbreds of domesticated maize chosen to represent the genetic diversity of maize germplasm, partial or nearly complete loss of the tandem DNA repeat CentC precedes 57 independent cenH3 relocation events that result in neocentromere formation. Chromosomal regions with newly acquired cenH3 are colonized by the centromere-specific retrotransposon CR2 at a rate that would result in centromere-sized CR2 clusters in 20,000-95,000 y. Three lines of evidence indicate that CentC loss is linked to inbreeding, including (i) CEN10 of temperate lineages, presumed to have experienced a genetic bottleneck, contain less CentC than their tropical relatives; (ii) strong selection for centromere-linked genes in domesticated maize reduced diversity at seven of the ten maize centromeres to only one or two postdomestication haplotypes; and (iii) the centromere with the largest number of haplotypes in domesticated maize (CEN7) has the highest CentC levels in nearly all domesticated lines. Rare recombinations introduced one (CEN2) or more (CEN5) alternate CEN haplotypes while retaining a single haplotype at domestication loci linked to these centromeres. Taken together, this evidence strongly suggests that inbreeding, favored by postdomestication selection for centromere-linked genes affecting key domestication or agricultural traits, drives replacement of the tandem centromere repeats in maize and other crop plants. Similar forces may act during speciation in natural systems.

Quantitative Profiling of Combinational K27/K36 Modifications on Histone H3 Variants in Mouse Organs.

The coexisting post-translational modifications (PTMs) on histone H3 N-terminal tails were known to crosstalk between each other, indicating their interdependency in the epigenetic regulation pathways. H3K36 methylation, an important activating mark, was recently reported to antagonize with PRC2-mediated H3K27 methylation with possible crosstalk mechanism during transcription regulation process. On the basis of our previous studies, we further integrated RP/HILIC liquid chromatography with MRM mass spectrometry to quantify histone PTMs from various mouse organs, especially the combinatorial K27/K36 marks for all three major histone H3 variants. Despite their subtle difference in physicochemical properties, we successfully obtained decent separation and high detection sensitivity for both histone H3.3 specific peptides and histone H3.1/3.2 specific peptides. In addition, the overall abundance of H3.3 can be quantified simultaneously. We applied this method to investigate the pattern of the combinatorial K27/K36 marks for all three major histone H3 variants across five mouse organs. Intriguing distribution differences were observed not only between different H3 variants but also between different organs. Our data shed the new insights into histone codes functions in epigenetic regulation during cell differentiation and developmental process.

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis.

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining

SummaryThe response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster.

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.

Investigating the Handedness Dynamics of Tetrasomes

Nucleosomes formed by DNA wrapping around histone octamers are the basic compaction unit of eukaryotic chromatin. Besides packaging DNA, they also play an essential role in genome regulation by controlling DNA accessibility. Their assembly follows a precise pathway in which first tetramers of H3 and H4 histones bind to DNA, forming tetrasomes, and then two dimers of H2A and H2B histones complete the formation of full nucleosomes. Here, we present our single-molecule studies on the assembly of tetrasomes by directly measuring the length, twist, and torque of single DNA molecules using Freely Orbiting Magnetic Tweezers (FOMT) and electro-Magnetic Torque Tweezers (eMTT). By this means, we have investigated both the replication-coupled histone H3.1 and its replication-independent variant histone H3.3. Our results indicate that tetrasomes exhibit a spontaneous flipping between a preferentially occupied left-handed state (ΔLk = −0.73) and a less populated right-handed state (ΔLk = +1.0), while weak torques convert them from one state into the other. In addition, the stepwise assembly of full nucleosomes suggests that tetrasomes undergoing changes in handedness remain viable intermediates. We further show how studies performed on tetrasomes that are artificially modified at their H3-H3 interface provide more insight into their handedness dynamics. Our findings reveal dynamical rearrangements of the nucleosomal structure that illustrate how chromatin could regulate DNA-supercoiling.

Histone Variant H3.3: A versatile H3 variant in health and in disease.

Histones are the main protein components of eukaryotic chromatin. Histone variants and histone modifications modulate chromatin structure, ensuring the precise operation of cellular processes associated with genomic DNA. H3.3, an ancient and conserved H3 variant, differs from its canonical H3 counterpart by only five amino acids, yet it plays essential and specific roles in gene transcription, DNA repair and in maintaining genome integrity. Here, we review the most recent insights into the functions of histone H3.3, and the involvement of its mutant forms in human diseases.

Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants

Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones.

In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry.

Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity, and availability of antihistone antibodies. Here, we describe a novel MALDI imaging MS based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available.