Histone H3 Variant Research Articles

Abstract IA14: The unique genomic landscape of pediatric high-grade glioma

Abstract Diffuse high-grade gliomas (HGGs) of childhood are a devastating spectrum of disease with no effective cures. The two-year survival for pediatric HGG ranges from 30 percent, for tumors arising in the cerebral cortex, to less than 10 percent for diffuse intrinsic pontine gliomas (DIPGs), which arise in the brainstem. Frequent recurrent somatic mutations in histone H3 distinguish the genetic drivers among pediatric and adult HGG. 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 and 22 percent of non-brainstem HGG, where they occur predominantly in thalamic and other midline tumors. Strikingly, heterozygous H3F3A mutations encoding histone H3.3 G34R or G34V occur in approximately 14 percent of non-brainstem HGG, and are associated predominantly with cortical tumors arising in older children through young adulthood, suggesting a distinct developmental origin compared to the majority of tumors harboring K27M mutations. Recurrent mutations of the BMP receptor ACVR1/ALK2 have not been reported outside the brainstem, and are further restricted to the youngest DIPG patients, highlighting critical connections between development and gliomagenesis. Experiments to model the connections between oncogenic activity and developmental context will be discussed. Citation Format: Jon D. Larson, Andre B. Silveira, Lawryn H. Kasper, Helen R. Russell, Chunxu Qu, Peter J. McKinnon, Suzanne J. Baker. The unique genomic landscape of pediatric high-grade glioma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA14.

Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns*

Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.

Abstract B06: Knock-down of H3.3 K27M mutant protein in DIPG cells delays, but does not abrogate, tumor growth in vivo

Abstract Genome-wide sequencing efforts of pediatric high grade gliomas (HGG) identified recurrent and hotspot mutations in the N-terminal regulatory tail of histone H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B/C). K27M histone mutations are mainly observed in midline tumors, including 78% of diffuse intrinsic pontine gliomas (DIPGs). By contrast, G34R/V mutations are predominantly restricted to 15% of the hemispheric tumours. This illustrates a highly specific selective pressure for each mutation in different cell types during brain development. The K27M mutation induces a dominant negative loss of trimethylated K27 (H3K27me3) marks in the bulk H3 pool. H3K27me3 interacts with Polycomb repressive complex 2 (PRC2) to repress transcription of target genes, a key process in the regulation and development of stem cells. Global gene expression analysis revealed that many significant differences between DIPGs (mostly K27M mutants) and non-brainstem pediatric HGG (mostly wild-type) are in genes that encode transcription factors and genes associated with developmental processes. Importantly, there was particular enrichment of genes exclusively expressed in distinct developing brain locations. Hence, it is challenging to define a clear gene expression signature associated with K27M mutations in DIPGs. In this study, in order to identify factors directly regulated by K27M, we aimed to knock-down the mutated protein in a DIPG-derived xenograft line. This H3.3 K27M line is maintained by successive implants of luciferase+/YFP+ tumor cells in the cortex of CD-1 nude immunocompromised mice. Resected tumor cells were transduced with lentiviral vectors expressing H3F3A specific shRNAs, selected with puromycin in neural stem cell culture conditions, and either kept under culture or re-implanted into mice. Cells in culture expressing any of three distinct shRNAs showed 92-96% H3F3A mRNA knock-down, 82-90% K27M protein knock-down and a 4-7 fold increase in H3K27me3 levels. Knock-down of H3F3A with any of three shRNAs in vivo significantly extended the time for tumor growth and the survival of xenografted mice. Notably, tumor growth was not completely abrogated. We observed an average of 79% H3F3A mRNA knock-down and increased levels of H3K27me3 by immunofluorescence in experimental tumors. Global gene expression profiling revealed modulation of a common subset of genes by all three shRNAs in vivo. We will present analysis of the gene expression changes associated with loss of the K27M mutation in DIPG cells. Overall, we show efficient knock-down of H3F3A in a H3.3 K27M mutant DIPG-derived xenograft line, leading to increased accumulation of H3K27me3, modulation of gene expression, and increased survival in vivo. Citation Format: André B. Silveira, Xiaoyan Zhu, Chunxu Qu, Suzanne Baker. Knock-down of H3.3 K27M mutant protein in DIPG cells delays, but does not abrogate, tumor growth in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B06.

Abstract A15: A human mini brain model of pediatric GBM

Abstract Glioblastoma (GBM) is the most devastating brain cancer with dismal prognosis both in adults and children and accounts for 16% of primary brain tumors. Despite aggressive therapeutic approaches, the majority of patients die within one year after diagnosis. Giving the limitation of current GBM mouse models, which do not reflect the biological properties of human tumors, in particular, tumor heterogeneity and pathogenesis, there is a growing need for new models. Current in vitro 3D models involve the use of hydrogel scaffolds and cell lines, which do not represent a viable developmental context where the tumor mass interacts with and invades the surrounding brain tissues. Starting with pluripotent human embryonic stem cells we reproduced a 3D tissue structure (so-called cerebral organoid) representing a developing human brain in vitro. We engineered these mini-brains to express oncogenic hits responsible for the derivation of tumors in pediatric GBM patients. Pediatric GBM is driven by a combination of epigenetic and genetic hits including the expression of a mutant histone H3 variant H3F3A (mutated at K27M or G34R) or a gain of function mutant isocitrate dehydrogenase IDH1R132H in combination with the loss of the histone remodeling factor ATRX and/or p53 tumor suppressor. We used a combination of these oncogenic hits to create a model of tumor engineering representing the first 3D human tissue model of GBM. This model combines the accurate multi-lineage differentiation and physiology of human brain with the easiness of manipulation of in vitro systems. H&E staining of the modified organoids shows remarkable dysplasia reminiscent of human primary glioma. Different genetic hits result in different type of dysplasia with IDH1R132H showing distinct morphology, suggestive of a broad range of applicability to model oncogenic-dependence in pediatric GBM. Cerebral organoids may also be used as a model to study tumor invasion starting with glioma stem-like cells (GSCs, which are believed to be the tumor-initiating cells in GBM). Indeed 923 GSCs (which are not invasive in mice brain) quickly and spontaneously (within few days) invade a co-cultured cerebral organoid. This study is the first to engineer a human CNS tumor in vitro. The model carries a tremendous potential to uncover the pathogenesis and initiation of brain cancer in a pediatric setting. Note: This abstract was not presented at the conference. Citation Format: Amin Ismail. A human mini brain model of pediatric GBM. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A15.

MLL5 Orchestrates a Cancer Self-Renewal State by Repressing the Histone Variant H3.3 and Globally Reorganizing Chromatin

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.

The molecular basis for centromere identity and function.

The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.

CBIO-35THE ROLE OF Spt6 IN GLIOBLASTOMA MAINTENANCE AND THERAPEUTIC RESISTANCE

Glioblastoma multiforme (GBM) is among the most lethal of solid cancers in adults. It is associated with a median survival of only approximately 15 months despite aggressive radio- and chemotherapies and recurrence is inevitable. Despite recent advances in our understanding of this deadly disease, the molecular mechanism and/or genes that cause high recurrence rates and treatment resistance in GBM are poorly understood. Histone chaperones affect the structure of the chromatin and expression of genes through interaction with histones and RNA polymerase II (PolII). Spt6 is a highly conserved transcription elongation factor and histone chaperone that has been found to counteract tri- methylation of the histone variant H3, at various lysines, an otherwise epigenetic mark which is associated with repression of transcription. We have identified a novel role of Spt6 in glioblastoma cells. Our data show that siRNA-mediated knockdown of Spt6 in GBM cells results in decreased proliferation rates and progression through the cell cycle, increased levels of DNA damage (measured by γH2AX) as well as delayed capacity for DNA repair after exposure to ionizing radiation. Altogether, our data imply an important role of Spt6 in glioblastoma maintenance and therapeutic resistance.

Atypical centromeres in plants-what they can tell us.

The centromere, visible as the primary constriction of condensed metaphase chromosomes, is a defined chromosomal locus essential for genome stability. It mediates transient assembly of a multi-protein complex, the kinetochore, which enables interaction with spindle fibers and thus faithful segregation of the genetic information during nuclear divisions. Centromeric DNA varies in extent and sequence composition among organisms, but a common feature of almost all active eukaryotic centromeres is the presence of the centromeric histone H3 variant cenH3 (a.k.a. CENP-A). These typical centromere features apply to most studied species. However, a number of species display “atypical” centromeres, such as holocentromeres (centromere extension along almost the entire chromatid length) or neocentromeres (ectopic centromere activity). In this review, we provide an overview of different atypical centromere types found in plants including holocentromeres, de novo formed centromeres and terminal neocentromeres as well as di-, tri- and metapolycentromeres (more than one centromere per chromosomes). We discuss their specific and common features and compare them to centromere types found in other eukaryotic species. We also highlight new insights into centromere biology gained in plants with atypical centromeres such as distinct mechanisms to define a holocentromere, specific adaptations in species with holocentromeres during meiosis or various scenarios leading to neocentromere formation.

DNA deletion as a mechanism for developmentally programmed centromere loss

A hallmark of active centromeres is the presence of the histone H3 variant CenH3 in the centromeric chromatin, which ensures faithful genome distribution at each cell division. A functional centromere can be inactivated, but the molecular mechanisms underlying the process of centromere inactivation remain largely unknown. Here, we describe the loss of CenH3 protein as part of a developmental program leading to the formation of the somatic nucleus in the eukaryote Paramecium. We identify two proteins whose depletion prevents developmental loss of CenH3: the domesticated transposase Pgm involved in the formation of DNA double strand cleavages and the Polycomb-like lysine methyltransferase Ezl1 necessary for trimethylation of histone H3 on lysine 9 and lysine 27. Taken together, our data support a model in which developmentally programmed centromere loss is caused by the elimination of DNA sequences associated with CenH3.

The histone chaperone DAXX maintains the structural organization of heterochromatin domains.

BackgroundThe death domain-associated protein (DAXX) collaborates with accessory proteins to deposit the histone variant H3.3 into mouse telomeric and pericentromeric repeat DNA. Pericentromeric repeats are the main genetic contributor to spatially discrete, compact, constitutive heterochromatic structures called chromocentres. Chromocentres are enriched in the H3K9me3 histone modification and serve as integral, functionally important components of nuclear organization. To date, the role of DAXX as an H3.3-specific histone chaperone has been investigated primarily using biochemical approaches which provide genome-wide views on cell populations and information on changes in local chromatin structures. However, the global chromatin and subnuclear reorganization events that coincide with these changes remain to be investigated.ResultsUsing electron spectroscopic imagine (ESI), a specialized form of energy-filtered transmission electron microscopy that allows us to visualize chromatin domains in situ with high contrast and spatial resolution, we show that in the absence of DAXX, H3K9me3-enriched domains are structurally altered and become uncoupled from major satellite DNA. In addition, the structural integrity of nucleoli and the organization of ribosomal DNA (rDNA) are disrupted. Moreover, the absence of DAXX leads to chromatin that is more sensitive, on a global level, to micrococcal nuclease digestion.ConclusionsWe identify a novel role of DAXX as a major regulator of subnuclear organization through the maintenance of the global heterochromatin structural landscape. As well, we show, for the first time, that the loss of a histone chaperone can have severe consequences for global nuclear organization.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0036-2) contains supplementary material, which is available to authorized users.

Functional interplay between histone H1 and HMG proteins in chromatin

The dynamic interaction of nucleosome binding proteins with their chromatin targets is an important element in regulating the structure and function of chromatin. Histone H1 variants and High Mobility Group (HMG) proteins are ubiquitously expressed in all vertebrate cells, bind dynamically to chromatin, and are known to affect chromatin condensation and the ability of regulatory factors to access their genomic binding sites. Here, we review the studies that focus on the interactions between H1 and HMGs and highlight the functional consequences of the interplay between these architectural chromatin binding proteins. H1 and HMG proteins are mobile molecules that bind to nucleosomes as members of a dynamic protein network. All HMGs compete with H1 for chromatin binding sites, in a dose dependent fashion, but each HMG family has specific effects on the interaction of H1 with chromatin. The interplay between H1 and HMGs affects chromatin organization and plays a role in epigenetic regulation. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.

DNA Topology and Global Architecture of Point Centromeres.

DNA is wrapped in a left-handed fashion around histone octasomes containing the centromeric histone H3 variant CENP-A. However, DNA topology studies have suggested that DNA is wrapped in a right-handed manner around the CENP-A nucleosome that occupies the yeast point centromere. Here, we determine the DNA linking number difference (ΔLk) stabilized by the yeast centromere and the contribution of the centromere determining elements (CDEI, CDEII, and CDEIII). We show that the intrinsic architecture of the yeast centromere stabilizes +0.6 units of ΔLk. This topology depends on the integrity of CDEII and CDEIII, but it is independent of cbf1 binding to CDEI and of the variable length of CDEII. These findings suggest that the interaction of the CBF3 complex with CDEIII and a distal CDEII segment configures a right-handed DNA loop that excludes CDEI. This loop is then occupied by a CENP-A histone complex, which does not have to be inherently right-handed.

Histone H3F3A and HIST1H3B K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes.

Diffuse intrinsic pontine glioma (DIPG) is the most severe paediatric solid tumour, with no significant therapeutic progress made in the past 50 years. Recent studies suggest that diffuse midline glioma, H3-K27M mutant, may comprise more than one biological entity. The aim of the study was to determine the clinical and biological variables that most impact their prognosis. Ninety-one patients with classically defined DIPG underwent a systematic stereotactic biopsy and were included in this observational retrospective study. Histone H3 genes mutations were assessed by immunochemistry and direct sequencing, whilst global gene expression profiling and chromosomal imbalances were determined by microarrays. A full description of the MRI findings at diagnosis and at relapse was integrated with the molecular profiling data and clinical outcome. All DIPG but one were found to harbour either a somatic H3-K27M mutation and/or loss of H3K27 trimethylation. We also discovered a novel K27M mutation in HIST2H3C, and a lysine-to-isoleucine substitution (K27I) in H3F3A, also creating a loss of trimethylation. Patients with tumours harbouring a K27M mutation in H3.3 (H3F3A) did not respond clinically to radiotherapy as well, relapsed significantly earlier and exhibited more metastatic recurrences than those in H3.1 (HIST1H3B/C). H3.3-K27M-mutated DIPG have a proneural/oligodendroglial phenotype and a pro-metastatic gene expression signature with PDGFRA activation, while H3.1-K27M-mutated tumours exhibit a mesenchymal/astrocytic phenotype and a pro-angiogenic/hypoxic signature supported by expression profiling and radiological findings. H3K27 alterations appear as the founding event in DIPG and the mutations in the two main histone H3 variants drive two distinct oncogenic programmes with potential specific therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1478-0) contains supplementary material, which is available to authorized users.

Structure and Stability of Telocentric Chromosomes in Wheat.

In most eukaryotes, centromeres assemble at a single location per chromosome. Naturally occurring telocentric chromosomes (telosomes) with a terminal centromere are rare but do exist. Telosomes arise through misdivision of centromeres in normal chromosomes, and their cytological stability depends on the structure of their kinetochores. The instability of telosomes may be attributed to the relative centromere size and the degree of completeness of their kinetochore. Here we test this hypothesis by analyzing the cytogenetic structure of wheat telosomes. We used a population of 80 telosomes arising from the misdivision of the 21 chromosomes of wheat that have shown stable inheritance over many generations. We analyzed centromere size by probing with the centromere-specific histone H3 variant, CENH3. Comparing the signal intensity for CENH3 between the intact chromosome and derived telosomes showed that the telosomes had approximately half the signal intensity compared to that of normal chromosomes. Immunofluorescence of CENH3 in a wheat stock with 28 telosomes revealed that none of the telosomes received a complete CENH3 domain. Some of the telosomes lacked centromere specific retrotransposons of wheat in the CENH3 domain, indicating that the stability of telosomes depends on the presence of CENH3 chromatin and not on the presence of CRW repeats. In addition to providing evidence for centromere shift, we also observed chromosomal aberrations including inversions and deletions in the short arm telosomes of double ditelosomic 1D and 6D stocks. The role of centromere-flanking, pericentromeric heterochromatin in mitosis is discussed with respect to genome/chromosome integrity.

Tissue-specific expression of histone H3 variants diversified after species separation.

BackgroundThe selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled.ResultsWe screened mouse, rat and human genomes by in silico hybridization using canonical histone sequences. In the mouse genome, we identified 14 uncharacterized H3 genes, among which 13 are similar to H3.3 and do not have human or rat counterparts, and one is similar to human testis-specific H3 variant, H3T/H3.4, and had a rat paralog. Although some of these genes were previously annotated as pseudogenes, their tissue-specific expression was confirmed by sequencing the 3′-UTR regions of the transcripts. Certain new variants were also detected at the protein level by mass spectrometry. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome-wide distributions of most variants were similar to that of H3.3. Moreover, forced expression of H3 variants in chromatin resulted in alternate gene expression patterns after cell differentiation.ConclusionsWe comprehensively identified and characterized novel mouse H3 variant genes that encoded highly conserved amino acid sequences compared to known histone H3. We speculated that the diversity of H3 variants acquired after species separation played a role in regulating tissue-specific gene expression in individual species. Their biological relevance and evolutionary aspect involving pseudogene diversification will be addressed by further functional analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0027-3) contains supplementary material, which is available to authorized users.

Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor with a median survival of 1 year after diagnosis. It has been reported recently that about 80% of DIPG cases and 70% of midline glioblastomas contain a mutation at one allele of the H3F3A gene (encoding histone H3 variant H3.3), replacing the lysine 27 with methionine (K27M). In order to facilitate diagnosis of DIPG patients, a quick and reliable method to identify the H3F3A K27M mutation is needed. Here, we describe a real-time PCR-based procedure involving a mutant-specific primer, a blocker oligonucleotide, and a reverse primer that can differentiate samples with H3F3A K27M mutation from those that do not. We first tested four different mutant-specific primers for their ability to selectively amplify H3F3A K27M-mutant allele and found that one primer amplified the mutant allele more efficiently than the rest. We then determined the optimal concentration of blocker oligo that significantly improved amplification of the H3F3A K27M-mutant allele. Using this optimized real-time PCR assay, we analyzed eleven samples, two of which containing H3F3A K27M mutation, and found that these two samples were differentially amplified from the nine others. In addition, we were able to discern the H3F3A K27M mutation in a newly obtained pediatric brainstem glioblastoma sample whose H3.3 status was not known previously, and in three other DIPG samples as well as paraffin embedded samples. These results demonstrate that we have developed a new reliable procedure for detecting the H3F3A K27M mutation in pediatric glioblastoma patient samples.

Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast.

Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibited unequal chromosome segregation, disrupted transcriptional silencing of centromeric chromatin, and reduced expression of Ams2, a GATA-factor that regulates localization of the centromere-specific histone H3 variant CENP-A. We found that overexpression of ams2+ could suppress the chromosome missegregation phenotype that arose in the hht2-S86AS87A mutant. We further demonstrate that centromeric localization of SpCENP-Acnp1-1 was significantly compromised in hht2-S86AS87A, suggesting synergism between histone H3 and the centromere-targeting domain of SpCENP-A. Taken together, our work presents evidence for an uncharacterized serine residue in fission yeast histone H3 that affects centromeric integrity via regulating the expression of the SpCENP-A-localizing Ams2 protein. [173/200 words]

A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis.

Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants.

Mis16 Independently Recognizes Histone H4 and the CENP-ACnp1-Specific Chaperone Scm3sp

CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-ACnp1-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-ACnp1 to centromeres. Although how Scm3sp specifically recognizes CENP-ACnp1 has been revealed recently, the recruiting mechanism of CENP-ACnp1 via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-ACnp1/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-ACnp1 specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-ACnp1/H4:Scm3sp into centromeres.

Stella controls chromocenter formation through regulation of Daxx expression in 2-cell embryos

In mammals, the structure of the pericentromeric region alters from a ring structure to a dot-like structure during the 2-cell stage. This structural alteration is termed chromocenter formation (CF) and is required for preimplantation development. Although reverse transcripts of major satellite repeats at pericentromeric regions are known to play roles in CF, its underlying mechanism is not fully understood. We previously reported that Stella (also known as PGC7 and Dppa3) deficiency led to developmental arrest at the preimplantation stage, accompanied by frequent chromosome segregation. In this study, we further investigated the effect of Stella deficiency on chromatin reorganization. The Stella-null embryos exhibited impaired CF and reduced expression of the reverse strand of major satellite repeats. In addition, the accumulation of H3.3, a histone H3 variant associated with transcriptional activation, at the pericentromeric regions and expression of the H3.3-specific chaperone Daxx were reduced in Stella-null embryos. These abnormalities were restored by the enforced expression of Daxx in Stella-null embryos. Thus, Stella controls the expression of Daxx and ensures chromatin reorganization in early embryos.