Facilitates Nucleosome Assembly Research Articles

Histone chaperones AtChz1A and AtChz1B are required for H2A.Z deposition and interact with the SWR1 chromatin-remodeling complex in Arabidopsis thaliana.

The histone variant H2A.Z plays key functions in transcription and genome stability in all eukaryotes ranging from yeast to human, but the molecular mechanisms by which H2A.Z is incorporated into chromatin remain largely obscure. Here, we characterized the two homologs of yeast Chaperone for H2A.Z-H2B (Chz1) in Arabidopsis thaliana, AtChz1A and AtChz1B. AtChz1A/AtChz1B were verified to bind to H2A.Z-H2B and facilitate nucleosome assembly invitro. Simultaneous knockdown of AtChz1A and AtChz1B, which exhibit redundant functions, led to a genome-wide reduction in H2A.Z and phenotypes similar to those of the H2A.Z-deficient mutant hta9-1hta11-2, including early flowering and abnormal flower morphologies. Interestingly, AtChz1A was found to physically interact with ACTIN-RELATED PROTEIN 6 (ARP6), an evolutionarily conserved subunit of the SWR1 chromatin-remodeling complex. Genetic interaction analyses showed that atchz1a-1atchz1b-1 was hypostatic to arp6-1. Consistently, genome-wide profiling analyses revealed partially overlapping genes and fewer misregulated genes and H2A.Z-reduced chromatin regions in atchz1a-1atchz1b-1 compared with arp6-1. Together, our results demonstrate that AtChz1A and AtChz1B act as histone chaperones to assist the deposition of H2A.Z into chromatin via interacting with SWR1, thereby playing critical roles in the transcription of genes involved in flowering and many other processes.

Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization.

Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite our increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. Here we identify four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. We show that TAP/p32 is required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, we identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells.

Two surfaces on the histone chaperone Rtt106 mediate histone binding, replication, and silencing.

The histone chaperone Rtt106 binds histone H3 acetylated at lysine 56 (H3K56ac) and facilitates nucleosome assembly during several molecular processes. Both the structural basis of this modification-specific recognition and how this recognition informs Rtt106 function are presently unclear. Guided by our crystal structure of Rtt106, we identified two regions on its double-pleckstrin homology domain architecture that mediated histone binding. When histone binding was compromised, Rtt106 localized properly to chromatin but failed to deliver H3K56ac, leading to replication and silencing defects. By mutating analogous regions in the structurally homologous chromatin-reorganizer Pob3, we revealed a conserved histone-binding function for a basic patch found on both proteins. In contrast, a loop connecting two β-strands was required for histone binding by Rtt106 but was dispensable for Pob3 function. Unlike Rtt106, Pob3 histone binding was modification-independent, implicating the loop of Rtt106 in H3K56ac-specific recognition in vivo. Our studies described the structural origins of Rtt106 function, identified a conserved histone-binding surface, and defined a critical role for Rtt106:H3K56ac-binding specificity in silencing and replication-coupled nucleosome turnover.

Towards a mechanism for histone chaperones

Histone chaperones can be broadly defined as histone-binding proteins that influence chromatin dynamics in an ATP-independent manner. Their existence reflects the importance of chromatin homeostasis and the unique and unusual biochemistry of the histone proteins. Histone supply and demand at chromatin is regulated by a network of structurally and functionally diverse histone chaperones. At the core of this network is a mechanistic variability that is only beginning to be appreciated. In this review, we highlight the challenges in determining histone chaperone mechanism and discuss possible mechanisms in the context of nucleosome thermodynamics. We discuss how histone chaperones prevent promiscuous histone interactions, and consider if this activity represents the full extent of histone chaperone function in governing chromatin dynamics. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional Output

The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation.

Solution Structure of Histone Chaperone ANP32B: Interaction with Core Histones H3–H4 through Its Acidic Concave Domain

Eukaryotic gene expression is regulated by histone deposition onto and eviction from nucleosomes, which are mediated by several chromatin-modulating factors. Among them, histone chaperones are key factors that facilitate nucleosome assembly. Acidic nuclear phosphoprotein 32B (ANP32B) belongs to the ANP32 family, which shares N-terminal leucine-rich repeats (LRRs) and a C-terminal variable anionic region. The C-terminal region functions as an inhibitor of histone acetylation, but the functional roles of the LRR domain in chromatin regulation have remained elusive. Here, we report that the LRR domain of ANP32B possesses histone chaperone activity and forms a curved structure with a parallel β-sheet on the concave side and mostly helical elements on the convex side. Our analyses revealed that the interaction of ANP32B with the core histones H3–H4 occurs on its concave side, and both the acidic and hydrophobic residues that compose the concave surface are critical for histone binding. These results provide a structural framework for understanding the functional mechanisms of acidic histone chaperones.

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A–H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A–H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A–H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A–H2B dimers onto DNA.

Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-gamma are required for cell viability. These observations highlighted the essential role of CAF-1-dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

The FK506-binding protein, Fpr4, is an acidic histone chaperone

Fpr4, a FK506-binding protein (FKBP), is a recently identified novel histone chaperone. How it interacts with histones and facilitates their deposition onto DNA, however, are not understood. Here, we report a functional analysis that shows Fpr4 forms complexes with histones and facilitates nucleosome assembly like previously characterized acidic histone chaperones. We also show that the chaperone activity of Fpr4 resides solely in an acidic domain, while the peptidylprolyl isomerase domain conserved among all FKBPs inhibits the chaperone activity. These observations argue that Fpr4, while unique structurally, deposits histones onto DNA for nucleosome assembly through the well-established mechanism shared by other chaperones.

Fpr4 Modulates the Structure of Core Histones

Fpr4 is a member of the FK506-binding protein (FKBP) family. Unlike the better studied FKBPs known as immunophilins that serve as intracellular receptors of immunosuppressants FK506 and rapamycin, Fpr4 contains an acidic domain in addition to the conserved FK506-binding domain that possesses peptidyl prolyl isomerase (PPIase) activity. Recently, it was reported that the Fpr4 is a novel histone chaperone with characteristics completely opposite to classic histone chaperones such as NAP1. Furthermore, the protein is shown to be required for gene silencing at the rDNA locus in the budding yeast Sacharomyces cerevisiae (Kuzuhara and Horikoshi, 2004, Nature Struc. & Mol. Biol.,11: 275–83). To gain insight into the function of this interesting protein, we have conducted a more detailed biochemical and genetic characterization of Fpr4. Our results show that Fpr4 facilitates nucleosome assembly in vitro in a process highly similar to that with NAP1. Furthermore, we found that the chaperone activity resides in the acidic domain and that the PPIase domain negatively regulates the chaperone activity of the protein. We have also that the PPIase domain contributes to the transcriptional repression at the rDNA locus, and is sufficient to repress transcription when tethered to the promoter of a reporter gene. Furthermore, we have demonstrated that the PPIase domain alters the topological structure of the histone H3–H4 tetramer in vitro. Taken together, our results suggest that Fpr4 is a protein that possesses a histone chaperone activity that facilitates nucleosome assembly and a peptidyl prolyl isomerase activity that may modulate the structure of the core histones. These activities, together, may impact chromatin structures and repress transcription at specific chromosome loci.

Structural Similarity between Histone Chaperone Cia1p/Asf1p and DNA-Binding Protein NF-κB

The structural relationships between histone-binding proteins and DNA-binding proteins are important, since nucleosome-interacting factors possess histone-binding and/or DNA-binding components. S. cerevisiae (Sc) Cia1p/Asf1p, a homologue of human CIA (CCG1-interacting factor A), is the most evolutionarily conserved histone chaperone, which facilitates nucleosome assembly by interacting with the nucleosome entry site of the core histones H3/H4. The crystal structure of the evolutionarily conserved domain (residues 1-169) of Cia1p (ScCia1p-DeltaC2) was determined at 2.95 A resolution. The refined model contains 166 residues in the asymmetric unit. The overall tertiary structure resembles a beta-sandwich fold, and belongs to the "switched" immunoglobulin class of proteins. The crystal structure suggests that ScCia1p-DeltaC2 is structurally related to the DNA-binding proteins, such as NF-kappaB and its family members. This is the first examination of the structural similarities between a histone chaperone and DNA-binding proteins. We discuss the possibilities that the strands beta3 and beta4, which possess highly electronegative surface potentials, are the important regions for the interaction with core histones, and that the histone chaperone ScCia1p/Asf1p and the DNA-binding protein NF-kappaB may have evolved from the same prototypal protein class.

Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast

In fission yeast, cia1+ is an essential gene that encodes a histone chaperone, a homologue of human CIA (CCG1-interacting factor A) and budding yeast Asf1p (anti-silencing function-1), which both facilitate nucleosome assembly by interacting with the core histones H3/H4. The conserved domain (residues 1-161) of the cia1+-encoded protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The protein was crystallized in the monoclinic space group C2, with unit-cell parameters a = 79.16, b = 40.53, c = 69.79 A, beta = 115.93 degrees and one molecule per asymmetric unit. The crystal diffracted to beyond 2.10 A resolution using synchrotron radiation.

Transcriptional Modification by a CASK-Interacting Nucleosome Assembly Protein

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization.

Nap1p is identified in mammalian cell extract by its intrinsic activity to facilitate nucleosome assembly in vitro in the physiological ionic condition. The homologous proteins are present in most eukaryotes, and their functional analyses in vitro have suggested that they are necessary to keep proper nucleosome structures in transcription and replication. This protein is also identified for its interaction with Clb2p in vitro. To address the function of Nap1p in vivo, we have surveyed for proteins to interact with Nap1p by two-hybrid system and obtained two genes, NBP1 and NBP2 (Nap1 Binding Protein 1 and 2). NBP1 is an essential gene and encodes a novel protein consisting of 319 amino acids, with a coiled-coil structure in the center of the predicted amino acid sequence. Several A-kinase dependent phosphorylation sites and Cdc28p kinase-dependent sites are also observed. By isolating the temperature-sensitive mutant, we demonstrate that the nuclear division at a non-permissive temperature is delayed and that the population of cells with a large bud carrying a single nucleus with a short spindle are increased. This mutant also confers resistance against benomyl, a microtubule-destabilizing agent. Judging from the green fluorescent protein (GFP) signal fused with Nbp1p, this protein localizes in the nucleus as one or two tiny dots.

Nucleosome Assembly on CTG Triplet Repeats

Expansion of CTG repeat sequences is associated with several human genetic diseases. We have examined the consequences of CTG repeat expansion for nucleosome assembly and positioning. Short CTG repeats are found within the most favored DNA sequences yet defined for nucleosome assembly. We find that as few as six CTG repeats will facilitate nucleosome assembly to a similar extent as the 50 or more repeats found in disease genes. Thus an increase in nucleosome stability on expansion of existing triplet repeats is unlikely to explain the acquisition of the disease phenotype. However, the CTG repeat sequence is efficiently wrapped around the histone octamer, preferring to associate with histones at the nucleosomal dyad. Thus short segments CTG repeat sequence will facilitate the assembly of a stable positioned nucleosome which might contribute to the expansion phenomenon and the functional organization of chromatin.

Identification and molecular cloning of yeast homolog of nucleosome assembly protein I which facilitates nucleosome assembly in vitro.

Yeast DNA coding for nucleosome assembly protein I (NAP-I), which facilitates nucleosome assembly in vitro at physiological ionic conditions, was cloned and its gene product was characterized. A monoclonal antibody against NAP-I (58 kDa) from human HeLa cells was used to screen a genomic library of Saccharomyces cerevisiae constructed into lambda gt11. A 60-kDa protein was detected by immunoblotting in the extracts of Escherichia coli lysogenized with a positive clone. The 60-kDa protein purified from the extracts had an activity equivalent to that of NAP-I from mouse and human cells. The amino acid sequence deduced from the gene coding for the yeast NAP-I defines a polypeptide of molecular mass 47,848 Da with three negatively charged regions. While the two regions contain 8 and 10 acidic amino acids out of 13 amino acid residues, the longest stretch has 15 glutamic and 13 aspartic acids out of 38 residues. These regions are probably involved in the interaction with histones. Proteins recognized by the anti-NAP-I antibody were also present in Xenopus oocytes and Drosophila cultured cells. Possible roles of NAP-I are discussed in relation to other nucleosome assembly proteins.

Multistep pathway for replication-dependent nucleosome assembly.

We have used cell-free DNA replication to study the relationship between DNA replication and chromatin assembly. As others have reported, we find that DNA replication facilitates nucleosome assembly. We show here that replication-dependent nucleosome assembly occurs in at least two steps. The first step requires replicating DNA; the second step occurs after replication has been completed and is promoted by a nuclear extract. Consistent with this multistep model, we observe that the replicated simian virus 40 minichromosome is organized into a repeating array of DNA-protein particles that are structurally distinct from mature nucleosomes. These particles may be precursors in a pathway of nucleosome assembly since in the second, replication-independent step the nuclear extract converts this nascent chromatin into nucleosomes.

H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro

Nucleosome formation has been studied in a system containing relaxed Col El DNA, core histones and an extract of Drosophila embryos. The formation of nucleosomes was established by the introduction of supercoils into DNA. The degree of DNA supercoiling was shown to be higher if nucleosomes were assem-bled in the presence of the H1 histone, polylysine ( M r 20000) or spermine. These agents do not stimulate relaxation and are the more effective the earlier they are added to the reaction. Thus, the H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro.

Purification and initial characterization of a protein which facilitates assembly of nucleosome-like structure from mammalian cells.

A protein, which facilitates assembly of a nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735-744]. The protein has been purified to approximately 80% from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex. From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed.

Nucleosome assembly.

Histones and DNA can spontaneously associate to form the nucleosome subunits of eukaryotic chromatin, but two proteins which occur in the eukaryotic nucleus can facilitate nucleosome assembly and greatly extend the conditions which permit assembly to occur. Recently several laboratories have reported new experimental systems which help to analyse, reconsruct and exploit the cellular mechanisms of chromatin assembly.