[show abstract][hide abstract] ABSTRACT: Centromeres are essential for ensuring proper chromosome segregation in eukaryotes. Their definition relies on the presence of a centromere-specific H3 histone variant CenH3, known as CENP-A in mammals. Its overexpression in aggressive cancers raises questions concerning its effect on chromatin dynamics and contribution to tumorigenesis. We find that CenH3 overexpression in human cells leads to ectopic enrichment at sites of active histone turnover involving a heterotypic tetramer containing CenH3-H4 with H3.3-H4. Ectopic localization of this particle depends on the H3.3 chaperone DAXX rather than the dedicated CenH3 chaperone HJURP. This aberrant nucleosome occludes CTCF binding and has a minor effect on gene expression. Cells overexpressing CenH3 are more tolerant of DNA damage. Both the survival advantage and CTCF occlusion in these cells are dependent on DAXX. Our findings illustrate how changes in histone variant levels can disrupt chromatin dynamics and suggests a possible mechanism for cell resistance to anticancer treatments.
[show abstract][hide abstract] ABSTRACT: The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.
[show abstract][hide abstract] ABSTRACT: BRCA1, BRCA2 and PALB2 are key players in cellular tolerance to chemotherapeutic agents including camptothecin, cisplatin and poly[ADP ribose]polymerase inhibitor. The N-terminal segment of BRCA2 interacts with PALB2, thus contributing to the formation of the BRCA1-PALB2-BRCA2 complex. To understand the role played by BRCA2 in this complex, we deleted its N-terminal segment and generated BRCA2N mutant cells. Although previous studies have suggested that BRCA1-PALB2 plays a role in the recruitment of BRCA2 to DNA-damage sites, BRCA2N mutant cells displayed a considerably milder phenotype than did BRCA2-/- null-deficient cells. We hypothesized that the DNA-binding domain of BRCA2 might compensate for a defect in BRCA2N that prevented stable interaction with PALB2. To test this hypothesis, we disrupted the DNA-binding domain of BRCA2 in wild-type and BRCA2N cells. Remarkably, although the resulting BRCA2DBD cells displayed a moderate phenotype, the BRCA2N+DBD cells displayed a very severe phenotype, as did the BRCA2-/- cells, suggesting that the N-terminal segment and the DNA-binding domain play a substantially overlapping role in the functionality of BRCA2. We also showed that the formation of both the BRCA1-PALB2-BRCA2 complex and the DNA-binding domain is required for efficient recruitment of BRCA2 to DNA-damage sites. Our study revealed the essential role played by both the BRCA1-PALB2-BRCA2 complex and the DNA-binding domain in the functionality of BRCA2, as each can compensate for the other in the recruitment of BRCA2 to DNA-damage sites. This knowledge adds to our ability to accurately predict the efficacy of anti-malignant therapies for patients carrying mutations in the BRCA2 gene.
[show abstract][hide abstract] ABSTRACT: The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP-A. In addition to CENP-A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP-T-W-S-X complex. However, it is unclear how the CENP-T-W-S-X complex associates with centromere chromatin. Here, we demonstrate that the CENP-T-W-S-X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP-T-W-S-X complex primarily binds to DNA as a (CENP-T-W-S-X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP-T-W-S-X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP-T or CENP-W, but not CENP-S or CENP-X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP-T-W-S-X complex. In summary, our work reveals the structural features and properties of the CENP-T-W-S-X complex for its localization to centromeres.
Nucleic Acids Research 11/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG1-3, which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.
Nucleic Acids Research 10/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.
Nucleic Acids Research 10/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA double-strand breaks (DSBs) are extremely dangerous DNA lesions. RAD51 plays a central role in homologous recombinational DSB repair, by facilitating the recombination of damaged DNA with intact DNA in eukaryotes. RAD51 accumulates at sites containing DNA damage to form nuclear foci. However, the mechanism of RAD51 accumulation at sites of DNA damage is still unclear. Posttranslational modifications of proteins, such as phosphorylation, acetylation and ubiquitination, play a role in the regulation of protein localization and dynamics. Recently, the covalent binding of small ubiquitin-like modifier (SUMO) proteins to target proteins, termed SUMOylation, at sites containing DNA damage has been determined to play a role in the regulation of the DNA damage response. Here we show that the SUMOylation E2, UBC9, and E3, PIAS1 and PIAS4 enzymes are required for RAD51 accretion at sites containing DNA damage in human cells. Moreover, we identified a SUMO-interacting motif (SIM) in RAD51, which is necessary for RAD51 accumulation at sites of DNA damage. These findings suggest that the SUMO-SIM system plays an important role in DNA repair, through the regulation of RAD51 dynamics.
Journal of Cell Science 09/2013; · 5.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.
[show abstract][hide abstract] ABSTRACT: It has been suggested that the hexasome, in which one of the H2A/H2B dimers is depleted from the canonical nucleosome core particle (NCP), is an essential intermediate during NCP assembly and disassembly, but little structural evidence for this exists. In this study, reconstituted products in a conventional NCP preparation were analyzed by native electrospray ionization mass spectrometry, and it was found that the hexasome, which migrated almost identically to the octasome NCP in native polyacrylamide gel electrophoresis, was produced simultaneously with the octasome NCP. This result might contribute to understanding the assembly and disassembly mechanism of NCPs.
[show abstract][hide abstract] ABSTRACT: The Epstein-Barr virus (EBV) genome is episomally maintained in latently infected cells. The viral protein EBNA1 is a bridging molecule that tethers EBV episomes to host mitotic chromosomes as well as to interphase chromatin. EBNA1 localizes to cellular chromosomes (chromatin) via its chromosome binding domains (CBDs), which are rich in glycine and arginine residues. However, the molecular mechanism by which the CBDs of EBNA1 attach to cellular chromatin is still under debate. Mutation analyses revealed that stepwise substitution of arginine residues within the CBD1 [amino acids (a.a.) 40-54] and CBD2 [a.a. 328-377] regions with alanines progressively impaired chromosome binding activity of EBNA1. The complete arginine-to-alanine substitutions within the CBD1 and 2 regions abolished the ability of EBNA1 to stably maintain EBV-derived oriP plasmids in dividing cells. Importantly, replacing the same arginines with lysines had minimal effect, if any, on chromosome binding of EBNA1 as well as on its ability to stably maintain oriP plasmids. Furthermore, a glycine-arginine rich peptide derived from the CBD1 region bound to reconstituted nucleosome core particles in vitro, as did a glycine-lysine rich peptide, while a glycine-alanine rich peptide did not. These results support the idea that the chromosome binding of EBNA1 is mediated by electrostatic interactions between the basic amino acids within the CBDs and negatively charged cellular chromatin.
Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Athe_0614 protein is a component of the extracellular proteins secreted by the anaerobic, extremely thermophilic and cellulolytic bacterium Caldicellulosiruptor bescii. The recombinant protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized using polyethylene glycol 2000 monomethyl ether as a precipitant. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.4, b = 42.2, c = 97.8 Å, β = 96.1°, and diffracted to 2.7 Å resolution using synchrotron radiation.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2013; 69(Pt 4):438-40. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: The minimum structural unit of chromatin is the nucleosome core particle (NCP), consisting of 146 bp of DNA wrapped around a histone octamer, which itself contains two H2A/H2B dimers and one (H3/H4)2 tetramer. These multimers possess functionally important tail regions that are intrinsically disordered. In order to elucidate the mechanisms behind NCP assembly and disassembly-processes which are highly related to gene expression-structural characterization of the H2A/H2B dimer and (H3/H4)2 tetramer will be of importance. In the present study, human histone multimers with disordered tail regions were characterized by electrospray ionization (ESI) ion mobility mass spectrometry (IM-MS) and molecular dynamics (MD) simulation. Experimentally obtained arrival times of these histone multimer ions showed rather wide distributions, implying that multiple conformers exist for each histone multimer in the gas phase. To examine their structures, MD simulations of the histone multimers were performed first in solution and then in vacuo at four temperatures, resulting in a variety of histone multimer structures. Theoretical collision cross-section (CCS) values calculated for the simulated structures revealed that structural models with smaller CCS values had more compact tail regions than those with larger CCS values. This implied that variation of the CCS values of the histone multimers were primarily due to the random behaviors of the tail regions in the gas phase. The combination of IM-MS and MD simulation enabled clear and comprehensive characterization of the gas-phase structures of histone multimers containing disordered tails.
[show abstract][hide abstract] ABSTRACT: In eukaryotes, transcription occurs in the chromatin context with the assistance of histone binding proteins, such as chromatin/nucleosome remodeling factors and histone chaperones. However, it is unclear how each remodeling factor or histone chaperone functions in transcription. Here, we identified a novel histone-binding protein, Spt2, in higher eukaryotes. Recombinant human Spt2 binds to histones and DNA, and promotes nucleosome assembly in vitro. Spt2 accumulates in nucleoli and interacts with RNA polymerase I in chicken DT40 cells, suggesting its involvement in ribosomal RNA transcription. Consistently, Spt2-deficient chicken DT40 cells are sensitive to RNA polymerase I inhibitors and exhibit decreased transcription activity, based on a transcription run-on assay. Domain analyses of Spt2 revealed that the C-terminal region, containing the region homologous to yeast Spt2, is responsible for histone binding, while the central region is essential for nucleolar localization and DNA binding. Based on these results, we conclude that vertebrate Spt2 is a novel histone chaperone with a separate DNA binding domain, facilitating ribosomal DNA transcription through chromatin remodeling during transcription.
Journal of Cell Science 02/2013; · 5.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: During meiosis, chromosomes adopt a bouquet arrangement, which is widely conserved among eukaryotes. This arrangement is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In Schizosaccharomyces pombe, the complex of Bqt1 and Bqt2 plays a key role in telomere clustering and the subsequent bouquet arrangement of chromosomes during early meiotic prophase. Bqt1 and Bqt2 are part of a multi-protein complex that mediates the attachment of the telomere to the nuclear membrane. However, the structural details of the complex are needed to clarify the mechanism of telomere clustering. To enable biophysical studies of Bqt1 and Bqt2, we established a purification procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, which is closely related to the S. pombe Bqt1-Bqt2 complex. A co-expression vector, in which one of the expressed subunits is fused to a removable SUMO tag, yielded high amounts of the proteins in the soluble fraction. The solubility of the Bqt1-Bqt2 complex after the removal of the SUMO tag was maintained by including CHAPS, a nondenaturing, zwitterionic detergent, in the purification buffers. These procedures enabled us to rapidly purify the stable Bqt1-Bqt2 complex. The co-purified Bqt1 and Bqt2 proteins formed a stable heterodimer, consistent with results from in vivo studies showing the requirement of both proteins for the bouquet arrangement. The expression and purification procedures established here will facilitate further biophysical studies of the Bqt1-Bqt2 complex.
Protein Expression and Purification 01/2013; · 1.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.
Nucleic Acids Research 01/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Histones are the protein components of the nucleosome, which forms the basic architecture of eukaryotic chromatin. Histones H2A, H2B, H3, and H4 are composed of two common regions, the "histone fold" and the "histone tail". Many efforts have been focused on the mechanisms by which the post-translational modifications of histone tails regulate the higher-order chromatin architecture. On the other hand, previous biochemical studies have suggested that histone tails also affect the structure and stability of the nucleosome core particle itself. However, the precise contributions of each histone tail are unclear. In the present study, we determined the crystal structures of four mutant nucleosomes, in which one of the four histones, H2A, H2B, H3, or H4, lacked the N-terminal tail. We found that the deletion of the H2B or H3 N-terminal tail affected histone-DNA interactions and substantially decreased nucleosome stability. These findings provide important information for understanding the complex roles of histone tails in regulating chromatin structure.
[show abstract][hide abstract] ABSTRACT: In higher eukaryotes, RAD51 functions as an essential protein in homologous recombination and recombinational repair of DNA double strand breaks. During these processes, RAD51 catalyzes homologous pairing between single-stranded DNA and double-stranded DNA. Japonica cultivars of rice (Oryza sativa) encode two RAD51 proteins, RAD51A1 and RAD51A2, whereas only one RAD51 exists in yeast and mammals. However, the functional differences between RAD51A1 and RAD51A2 have not been elucidated, because their biochemical properties have not been characterized. In the present study, we purified RAD51A1 and RAD51A2, and found that RAD51A2 robustly promotes homologous pairing in vitro. RAD51A1 also possesses homologous-pairing activity, but it is only about 10% of the RAD51A2 activity. Both RAD51A1 and RAD51A2 bind to ssDNA and dsDNA, and their DNA binding strictly requires ATP, which modulates the polymer formation activities of RAD51A1 and RAD51A2. These findings suggest that although both RAD51A1 and RAD51A2 have the potential to catalyze homologous pairing, RAD51A2 may be the major recombinase in rice.
PLoS ONE 01/2013; 8(10):e75451. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.
[show abstract][hide abstract] ABSTRACT: The nucleosome is the basic repeating unit of chromatin. During the nucleosome assembly process, DNA is wrapped around two H3-H4 dimers, followed by the inclusion of two H2A-H2B dimers. The H3-H4 dimers provide the fundamental architecture of the nucleosome. Many non-allelic variants have been found for H3, but not for H4, suggesting that the functions of chromatin domains may, at least in part, be dictated by the specific H3 variant that is incorporated. A prominent example is the centromeric H3 variant, CENP-A, which specifies the function of centromeres in chromosomes. In this review, we survey the current progress in the studies of nucleosomes containing H3 variants, and discuss their implications for the architecture and dynamics of chromatin domains.
Current Opinion in Structural Biology 12/2012; · 8.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.
The EMBO Journal 07/2012; 31(17):3524-36. · 9.82 Impact Factor