Akihisa Osakabe

Crystal structure of the human centromeric nucleosome containing CENP-A

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T

A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for meiosis and/or spermatogenesis. In the present study, we found that the nucleosome containing human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome containing H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central α2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences observed between H3T and H3.1. A mutational analysis revealed that these residues are responsible for the reduced stability of the H3T-containing nucleosome. These physical and structural properties of the H3T-containing nucleosome may provide the basis of chromatin reorganization during spermatogenesis.

Structures of human nucleosomes containing major histone H3 variants

The nucleosome is the fundamental repeating unit of chromatin, via which genomic DNA is packaged into the nucleus in eukaryotes. In the nucleosome, two copies of each core histone, H2A, H2B, H3 and H4, form a histone octamer which wraps 146 base pairs of DNA around itself. All of the core histones except for histone H4 have nonallelic isoforms called histone variants. In humans, eight histone H3 variants, H3.1, H3.2, H3.3, H3T, H3.5, H3.X, H3.Y and CENP-A, have been reported to date. Previous studies have suggested that histone H3 variants possess distinct functions in the formation of specific chromosome regions and/or in the regulation of transcription and replication. H3.1, H3.2 and H3.3 are the most abundant H3 variants. Here, crystal structures of human nucleosomes containing either H3.2 or H3.3 have been solved. The structures were essentially the same as that of the H3.1 nucleosome. Since the amino-acid residues specific for H3.2 and H3.3 are located on the accessible surface of the H3/H4 tetramer, they may be potential interaction sites for H3.2- and H3.3-specific chaperones.

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

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.

Nucleosome formation activity of human somatic nuclear autoantigenic sperm protein (sNASP).

NASP (nuclear autoantigenic sperm protein) is a member of the N1/N2 family, which is widely conserved among eukaryotes. Human NASP reportedly prefers to bind to histones H3·H4 and the linker histone H1, as compared with H2A·H2B, and is anticipated to function as an H3·H4 chaperone for nucleosome assembly. However, the direct nucleosome assembly activity of human NASP has not been reported so far. In humans, two spliced isoforms, somatic and testicular NASPs (sNASP and tNASP, respectively) were identified. In the present study we purified human sNASP and found that sNASP efficiently promoted the assembly of nucleosomes containing the conventional H3.1, H3.2, H3.3, or centromere-specific CENP-A. On the other hand, sNASP inefficiently promoted nucleosome assembly with H3T, a testis-specific H3 variant. Mutational analyses revealed that the Met-71 residue of H3T is responsible for this inefficient nucleosome formation by sNASP. Tetrasomes, composed of the H3·H4 tetramer and DNA without H2A·H2B, were efficiently formed by the sNASP-mediated nucleosome-assembly reaction. A deletion analysis of sNASP revealed that the central region, amino acid residues 26–325, of sNASP is responsible for nucleosome assembly in vitro. These experiments are the first demonstration that human NASP directly promotes nucleosome assembly and provide compelling evidence that sNASP is a bona fide histone chaperone for H3·H4.

Contribution of histone N-terminal tails to the structure and stability of nucleosomes

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.

The centromeric nucleosome-like CENP–T–W–S–X complex induces positive supercoils into DNA

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.

Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin

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.

Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2

The crystal structures of human nucleosomes containing H2A.Z.1 and H2A.Z.2 have been determined. Structural polymorphisms were found in the L1 loop regions of H2A.Z.1 and H2A.Z.2 in the nucleosomes that are likely to be caused by their flexible nature.

Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.