H3-H4 Dimer Research Articles

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome byengaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Understanding HAT1: A Comprehensive Review of Noncanonical Roles and Connection with Disease.

Histone acetylation plays a vital role in organizing chromatin, regulating gene expression and controlling the cell cycle. The first histone acetyltransferase to be identified was histone acetyltransferase 1 (HAT1), but it remains one of the least understood acetyltransferases. HAT1 catalyzes the acetylation of newly synthesized H4 and, to a lesser extent, H2A in the cytoplasm. However, 20 min after assembly, histones lose acetylation marks. Moreover, new noncanonical functions have been described for HAT1, revealing its complexity and complicating the understanding of its functions. Recently discovered roles include facilitating the translocation of the H3H4 dimer into the nucleus, increasing the stability of the DNA replication fork, replication-coupled chromatin assembly, coordination of histone production, DNA damage repair, telomeric silencing, epigenetic regulation of nuclear lamina-associated heterochromatin, regulation of the NF-κB response, succinyl transferase activity and mitochondrial protein acetylation. In addition, the functions and expression levels of HAT1 have been linked to many diseases, such as many types of cancer, viral infections (hepatitis B virus, human immunodeficiency virus and viperin synthesis) and inflammatory diseases (chronic obstructive pulmonary disease, atherosclerosis and ischemic stroke). The collective data reveal that HAT1 is a promising therapeutic target, and novel therapeutic approaches, such as RNA interference and the use of aptamers, bisubstrate inhibitors and small-molecule inhibitors, are being evaluated at the preclinical level.

Cryo–electron microscopy structure of the H3-H4 octasome: A nucleosome-like particle without histones H2A and H2B

The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo-electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4' four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone-histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.

Structure of IMPORTIN-4 bound to the H3–H4–ASF1 histone–histone chaperone complex

IMPORTIN-4, the primary nuclear import receptor of core histones H3 and H4, binds the H3-H4 dimer and histone chaperone ASF1 prior to nuclear import. However, how H3-H3-ASF1 is recognized for transport cannot be explained by available crystal structures of IMPORTIN-4-histone tail peptide complexes. Our 3.5-Å IMPORTIN-4-H3-H4-ASF1 cryoelectron microscopy structure reveals the full nuclear import complex and shows a binding mode different from suggested by previous structures. The N-terminal half of IMPORTIN-4 clamps the globular H3-H4 domain and H3 αN helix, while its C-terminal half binds the H3 N-terminal tail weakly; tail contribution to binding energy is negligible. ASF1 binds H3-H4 without contacting IMPORTIN-4. Together, ASF1 and IMPORTIN-4 shield nucleosomal H3-H4 surfaces to chaperone and import it into the nucleus where RanGTP binds IMPORTIN-4, causing large conformational changes to release H3-H4-ASF1. This work explains how full-length H3-H4 binds IMPORTIN-4 in the cytoplasm and how it is released in the nucleus.

A specific role for importin-5 and NASP in the import and nuclear hand-off of monomeric H3.

Core histones package chromosomal DNA and regulate genomic transactions, with their nuclear import and deposition involving importin-β proteins and a dedicated repertoire of histone chaperones. Previously, a histone H3-H4 dimer has been isolated bound to importin-4 (Imp4) and the chaperone ASF1, suggesting that H3 and H4 fold together in the cytoplasm before nuclear import. However, other studies have shown the existence of monomeric H3 in the nucleus, indicating a post-import folding pathway. Here, we report that the predominant importin associated with cytoplasmic H3 is importin-5 (Imp5), which hands off its monomeric cargo to nuclear sNASP. Imp5, in contrast to Imp4, binds to both H3 and H4 containing constitutively monomeric mutations and binds to newly synthesised, monomeric H3 tethered in the cytoplasm. Constitutively monomeric H3 retains its interaction with NASP, whereas monomeric H4 retains interactions specifically with HAT1 and RBBP7. High-resolution separation of NASP interactors shows the 's' isoform but not the 't' isoform associates with monomeric H3, whilst both isoforms associate with H3-H4 dimers in at least three discrete multi-chaperoning complexes. In vitro binding experiments show mutual exclusivity between sNASP and Imp5 in binding H3, suggesting direct competition for interaction sites, with the GTP-bound form of Ran required for histone transfer. Finally, using pulse-chase analysis, we show that cytoplasm-tethered histones do not interact with endogenous NASP until they reach the nucleus, whereupon they bind rapidly. We propose an Imp5-specific import pathway for monomeric H3 that hands off to sNASP in the nucleus, with a parallel H4 pathway involving Imp5 and the HAT1-RBBP7 complex, followed by nuclear folding and hand-off to deposition factors.

Epstein-Barr Virus Tegument Protein BKRF4 is a Histone Chaperone

Histone chaperones, which constitute an interaction and functional network involved in all aspects of histone metabolism, have to date been identified only in eukaryotes. The Epstein-Barr virus tegument protein BKRF4 is a histone-binding protein that engages histones H2A–H2B and H3–H4, and cellular chromatin, inhibiting the host DNA damage response. Here, we identified BKRF4 as a bona fide viral histone chaperone whose histone-binding domain (HBD) forms a co-chaperone complex with the human histone chaperone ASF1 in vitro. We determined the crystal structures of the quaternary complex of the BKRF4 HBD with human H3–H4 dimer and the histone chaperone ASF1b and the ternary complex of the BKRF4 HBD with human H2A–H2B dimer. Through structural and biochemical studies, we elucidated the molecular basis for H3–H4 and H2A–H2B recognition by BKRF4. We also revealed two conserved motifs, D/EL and DEF/Y/W, within the BKRF4 HBD, which may represent common motifs through which histone chaperones target H3–H4 and H2A–H2B, respectively. In conclusion, our results identify BKRF4 as a histone chaperone encoded by the Epstein-Barr virus, representing a typical histone chaperone found in a non-eukaryote. We envision that more histone chaperones await identification and characterization in DNA viruses and even archaea.

DIPG-20. Copper chelation therapy targets S-adenosylmethionine (SAM) metabolism and epigenetic regulators in diffuse intrinsic pontine glioma (DIPG)

DIPG is an incurable pediatric brain cancer of the ventral pons characterized by its complex epigenetic profile. Up to 81% of patients present with mutation H3K27M, resulting in the global reduction of H3K27 trimethylation and increased H3K27 acetylation, deregulating gene expression through an aberrant pattern of epigenetic modification. These alterations affect many cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper is highly abundant in the pons and is essential for normal brain function and development. However, excess copper accumulation is implicated in several neurological diseases and cancers. Incidentally, recent investigations have indicated the H3-H4 dimer can interact with copper acting as a reductase enzyme. Copper chelation therapy is clinically approved for pediatric patients with Wilson’s Disease, improving their neurological symptoms, and is being trialed in several cancers. We therefore hypothesized copper chelation may represent an effective therapeutic strategy for DIPG. Copper chelator tetraethylenepentamine (TEPA) decreased cell growth and induced apoptosis in DIPG cell lines. To understand these results, unbiased RNA-seq and metabolomics analyses were performed, revealing downregulation of EZH2, DNMT1 and DNMT3B, upregulation of KDM6B and the disruption of key enzymes in the S-adenosylmethionine (SAM)-cycle. Importantly, TEPA downregulated SAM, which donates methyl groups for methylation, S-adenosylhomocysteine, its post-methylation product and α-ketoglutarate, a co-factor for KDM6B. Western blots confirmed the reduced expression of EZH2, DNMT1 and DNMT3B, with further blots examining the chromatin cell fraction revealing modulation of H3K27 trimethylation through copper or TEPA stimulation, and reduction of H3K27 acetylation by TEPA. Importantly, in vitro combinations with Panobinostat were synergistic, while in vivo investigations demonstrated TEPA improved survival in an orthotopic patient derived xenograft (PDX) model, showing complete tumor regression in 25% of treated mice. This study indicates a novel use for copper chelators as epigenetic drugs, and their potential as therapeutics for DIPG.

Distinct structural groups of histone H3 and H4 residues have divergent effects on chronological lifespan in Saccharomyces cerevisiae.

We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the transcriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism.

Spn1 and Its Dynamic Interactions with Spt6, Histones and Nucleosomes

Histone chaperones facilitate the assembly and disassembly of nucleosomes and regulate DNA accessibility for critical cellular processes. Spn1 is an essential, highly conserved histone chaperone that functions in transcription initiation and elongation in a chromatin context. Here we demonstrate that Spn1 binds H3-H4 with low nanomolar affinity, residues 85–99 within the acidic N-terminal region of Spn1 are required for H3-H4 binding, and Spn1 binding to H3-H4 dimers does not impede (H3-H4)2 tetramer formation. Previous work has shown the central region of Spn1 (residues 141–305) is important for interaction with Spt6, another conserved and essential histone chaperone. We show that the C-terminal region of Spn1 also contributes to Spt6 binding and is critical for Spn1 binding to nucleosomes. We also show Spt6 preferentially binds H3-H4 tetramers and Spt6 competes with nucleosomes for Spn1 binding. Combined with previous results, this indicates the Spn1-Spt6 complex does not bind nucleosomes. In contrast to nucleosome binding, we found that the Spn1-Spt6 complex can bind H3-H4 dimers and tetramers and H2A-H2B to form ternary complexes. These important results provide new information about the functions of Spn1, Spt6, and the Spn1-Spt6 complex, two essential and highly conserved histone chaperones.

NASP maintains histone H3–H4 homeostasis through two distinct H3 binding modes

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3–H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3–H4 supply. We determine the structures of a sNASP dimer, a complex of a sNASP dimer with two H3 α3 peptides, and the sNASP–H3–H4–ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 α3 helix and the H3 αN region, respectively. Functional studies demonstrate the H3 αN-interaction represents the major binding mode of NASP in cells and shielding of the H3 αN region by NASP is essential in maintaining the H3–H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3–H4 chaperone in guarding histone homeostasis.

Mechanistic insights into histone deposition and nucleosome assembly by the chromatin assembly factor-1.

Eukaryotic chromatin is a highly dynamic structure with essential roles in virtually all DNA-dependent cellular processes. Nucleosomes are a barrier to DNA access, and during DNA replication, they are disassembled ahead of the replication machinery (the replisome) and reassembled following its passage. The Histone chaperone Chromatin Assembly Factor-1 (CAF-1) interacts with the replisome and deposits H3–H4 directly onto newly synthesized DNA. Therefore, CAF-1 is important for the establishment and propagation of chromatin structure. The molecular mechanism by which CAF-1 mediates H3–H4 deposition has remained unclear. However, recent studies have revealed new insights into the architecture and stoichiometry of the trimeric CAF-1 complex and how it interacts with and deposits H3–H4 onto substrate DNA. The CAF-1 trimer binds to a single H3–H4 dimer, which induces a conformational rearrangement in CAF-1 promoting its interaction with substrate DNA. Two CAF-1•H3–H4 complexes co-associate on nucleosome-free DNA depositing (H3–H4)2 tetramers in the first step of nucleosome assembly. Here, we review the progress made in our understanding of CAF-1 structure, mechanism of action, and how CAF-1 contributes to chromatin dynamics during DNA replication.

Mathematical modeling of the temperature effect on the character of linking between monomeric proteins in aqueous solutions

The mathematical model was developed for taking into account the influence of the temperature of an aqueous salt-free solution on the character of dimer formation by different sections of small proteins: H2A–H2B and H3–H4 dimers, and the effect of temperature on various sections of the Bcl-xl protein were studied. The analysis of the numerical calculations obtained in the course of the developed mathematical model revealed a different behavior of the histone dimers H2B-H2A and H3–H4, as well as the Bcl-xl\(_{(1-212)}\)–Bcl-xl\(_{(1-212)}\), Bcl-xl–Bcl-xl, Bcl-xl\(_{(1-212)}\)–Bcl-xl\(_{(213-233)}\), Bcl-xl\(_{(213-233)}\)–Bcl-xl\(_{(213-233)}\) with an increase of temperature from 20 \(^\circ \)C to 40 \(^\circ \)C, as well as the contribution of different sections of the Bcl-xl protein to the formation of the biological complex.

Yeast CAF-1 assembles histone (H3-H4) 2 tetramers prior to DNA deposition.

Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)2 tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)2 tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)2 tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.

DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication.

Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4)2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.

Maintenance of Heterochromatin by the Large Subunit of the CAF-1 Replication-Coupled Histone Chaperone Requires Its Interaction with HP1a Through a Conserved Motif.

In eukaryotic cells, the organization of genomic DNA into chromatin regulates many biological processes, from the control of gene expression to the regulation of chromosome segregation. The proper maintenance of this structure upon cell division is therefore of prime importance during development for the maintenance of cell identity and genome stability. The chromatin assembly factor 1 (CAF-1) is involved in the assembly of H3-H4 histone dimers on newly synthesized DNA and in the maintenance of a higher order structure, the heterochromatin, through an interaction of its large subunit with the heterochromatin protein HP1a. We identify here a conserved domain in the large subunit of the CAF-1 complex required for its interaction with HP1a in the Drosophila fruit fly. Functional analysis reveals that this domain is dispensable for viability but participates in two processes involving heterochromatin: position-effect variegation and long range chromosomal interactions during meiotic prophase. Importantly, the identification in the large subunit of CAF-1 of a domain required for its interaction with HP1 allows the separation of its functions in heterochromatin-related processes from its function in the assembly of H3-H4 dimers onto newly synthesized DNA.

SNASP and ASF1A function through both competitive and compatible modes of histone binding.

Histone chaperones are proteins that interact with histones to regulate the thermodynamic process of nucleosome assembly. sNASP and ASF1 are conserved histone chaperones that interact with histones H3 and H4 and are found in a multi-chaperoning complex in vivo. Previously we identified a short peptide motif within H3 that binds to the TPR domain of sNASP with nanomolar affinity. Interestingly, this peptide motif is sequestered within the known ASF1–H3–H4 interface, raising the question of how these two proteins are found in complex together with histones when they share the same binding site. Here, we show that sNASP contains at least two additional histone interaction sites that, unlike the TPR–H3 peptide interaction, are compatible with ASF1A binding. These surfaces allow ASF1A to form a quaternary complex with both sNASP and H3–H4. Furthermore, we demonstrate that sNASP makes a specific complex with H3 on its own in vitro, but not with H4, suggesting that it could work upstream of ASF1A. Further, we show that sNASP and ASF1A are capable of folding an H3–H4 dimer in vitro under native conditions. These findings reveal a network of binding events that may promote the entry of histones H3 and H4 into the nucleosome assembly pathway.

A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork.

MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.

Characterization of the Pneumocystis carinii Histone Acetyltransferase Chaperone Proteins PcAsf1 and PcVps75

Rtt109 is a lysine acetyltransferase that acetylates histone H3 at lysine 56 (H3K56) in fungi. This acetylation event is important for proper DNA replication and repair to occur. Efficient Rtt109 acetyltransferase activity also requires a histone chaperone, vacuolar protein sorting 75 (Vps75), as well as the major chaperone of the H3-H4 dimer, anti-silencing factor 1 (Asf1). Little is known about the role of these proteins in the opportunistic fungal pathogen Pneumocystis carinii. To investigate the functions of Asf1 and Vps75 in Pneumocystis carinii, we cloned and characterized both of these genes. Here, we demonstrate that both genes, P. carinii asf1 (Pcasf1) and Pcvps75, function in a fashion analogous to their Saccharomyces cerevisiae counterparts. We demonstrate that both P. carinii Asf1 (PcAsf1) and PcVps75 can bind histones. Furthermore, when Pcasf1 is expressed heterologously in S. cerevisiae asf1Δ cells, PcAsf1 can restore full H3 lysine acetylation. We further demonstrated that the Pcasf1 cDNA expressed in asf1Δ S. cerevisiae cells can restore growth to wild-type levels in the presence of genotoxic agents that block DNA replication. Lastly, we observed that purified PcAsf1 and PcVps75 proteins enhance the ability of PcRtt109 to acetylate histone H3-H4 tetramers. Together, our results indicate that the functions of the Rtt109-Asf1-Vps75 complex in the acetylation of histone H3 lysine 56 and in DNA damage response are present in P. carinii DNA and cell cycle progression.

Current progress on structural studies of nucleosomes containing histone H3 variants

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.