Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics

Background  

RBBP6 is a 250 kDa splicing-associated protein that has been identified as an E3 ligase due to the presence of a RING finger domain. In humans and mice it interacts with both p53 and Rb, and plays a role in the induction of apoptosis and regulation of the cell cycle. RBBP6 has recently been shown to be highly up-regulated in oesophageal cancer, and to be a promising target for immunotherapy against the disease.     

Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights

Asf1 is a histone chaperone that favors histone H3/H4 assembly and disassembly. We solved the structure of the conserved domain of human ASF1A in complex with the C-terminal helix of histone H3 using nuclear magnetic resonance spectroscopy. This structure is fully compatible with an association of ASF1 with the heterodimeric form of histones H3/H4. In our model, ASF1 substitutes for the second H3/H4 heterodimer that is normally found in heterotetrameric H3/H4 complexes. This result constitutes an essential step in the fundamental understanding of the mechanisms of nucleosome assembly by histone chaperones. Point mutations that perturb the Asf1/histone interface were designed from the structure. The decreased binding affinity of the Asf1-H3/H4 complex correlates with decreased levels of H3-K56 acetylation and phenotypic defects in vivo.     

The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4-DNA complexes

The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be the first and the last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) is a chaperone of the H3/H4 dimer that functions in both of these processes. However, little is known about the thermodynamics of chaperone–histone interactions or the direct role of Asf1 in the formation or disassembly of histone–DNA complexes. Here, we show that Saccharomyces cerevisiae Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone–DNA interactions through the formation of disomes. However, Asf1 was unable to disengage histones from DNA for tetrasomes formed with H3/H4 and strong nucleosome positioning DNA sequences or tetrasomes weakened by mutant (H3K56Q/H4) histones or non-positioning DNA sequences. Furthermore, Asf1 did not associate with preformed tetrasomes. These results are consistent with the measured affinity of Asf1 for H3/H4 dimers of 2.5 nM, which is weaker than the association of H3/H4 for DNA. These studies support a mechanism by which Asf1 aids H3/H4 deposition onto DNA but suggest that additional factors or post-translational modifications are required for Asf1 to remove H3/H4 from tetrasome intermediates in chromatin.     

Chz1, a nuclear chaperone for histone H2AZ

The histone variant H2AZ marks nucleosomes flanking the promoters of most genes of budding yeast. The incorporation of H2AZ into chromatin is dependent on the SWR1 complex, which catalyses the replacement of conventional histone H2A with H2AZ. In cells, the pool of unincorporated histone H2AZ has previously been found in association with Nap1, a chaperone for conventional histone H2A-H2B. Here, we report the discovery of Chz1, a histone chaperone that has preference for H2AZ and can also deliver a source of the histone variant for SWR1-dependent histone replacement. Bacterially expressed Chz1 forms a heterotrimer with H2AZ-H2B, stabilizing the association of the histone dimer. We have identified a conserved motif important for histone variant recognition within the H2AZ-interacting domain of Chz1. The presence of this motif in other metazoan proteins suggests that H2AZ-specific chaperones may be widely conserved.     

Gene regulation: implications of histone dispersal patterns for epigenetics

Histones are widely believed to carry regulatory information across cell generations. A recent study suggests limits to this model by measuring dispersal of ancestral histones in yeast.     

Structure, chemical modification and interaction of histone H1 with chromatin components

Structure, chemical modification, and interaction of histone H1 and its individual fragments with DNA and structural elements of chromatin are considered. Special attention is paid to phosphorylation of histone H1 molecules. Recent data concerning localization and mobility of histone H1 in chromatin as well as mechanisms of nucleosomal chain condensation are reviewed.     

Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase

Methylation of Lys79 on histone H3 by Dot1p is important for gene silencing. The elongated structure of the conserved core of yeast Dot1p contains an N-terminal helical domain and a seven-stranded catalytic domain that harbors the binding site for the methyl-donor and an active site pocket sided with conserved hydrophobic residues. The S-adenosyl-L-homocysteine exhibits an extended conformation distinct from the folded conformation observed in structures of SET domain histone lysine methyltransferases. A catalytic asparagine (Asn479), located at the bottom of the active site pocket, suggests a mechanism similar to that employed for amino methylation in DNA and protein glutamine methylation. The acidic, concave cleft between the two domains contains two basic residue binding pockets that could accommodate the outwardly protruding basic side chains around Lys79 of histone H3 on the disk-like nucleosome surface. Biochemical studies suggest that recombinant Dot1 proteins are active on recombinant nucleosomes, free of any modifications.     

Importance of electrostatic interactions in the association of intrinsically disordered histone chaperone Chz1 and histone H2A.Z-H2B

Histone chaperones facilitate assembly and disassembly of nucleosomes. Understanding the process of how histone chaperones associate and dissociate from the histones can help clarify their roles in chromosome metabolism. Some histone chaperones are intrinsically disordered proteins (IDPs). Recent studies of IDPs revealed that the recognition of the biomolecules is realized by the flexibility and dynamics, challenging the century-old structure-function paradigm. Here we investigate the binding between intrinsically disordered chaperone Chz1 and histone variant H2A.Z-H2B by developing a structure-based coarse-grained model, in which Debye-Hückel model is implemented for describing electrostatic interactions due to highly charged characteristic of Chz1 and H2A.Z-H2B. We find that major structural changes of Chz1 only occur after the rate-limiting electrostatic dominant transition state and Chz1 undergoes folding coupled binding through two parallel pathways. Interestingly, although the electrostatic interactions stabilize bound complex and facilitate the recognition at first stage, the rate for formation of the complex is not always accelerated due to slow escape of conformations with non-native electrostatic interactions at low salt concentrations. Our studies provide an ionic-strength-controlled binding/folding mechanism, leading to a cooperative mechanism of "local collapse or trapping" and "fly-casting" together and a new understanding of the roles of electrostatic interactions in IDPs' binding.     

Histone H1--DNA interaction. On the mechanism of DNA strands crosslinking by histone H1.

Crosslinking of DNA fibers by histone H1 or phosphorylated on Ser-37 histone H1, and by the individual fragments of the H1 polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of H1.DNA complexes in vitro is apparently due to both specific histone-DNA interactions with the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of shrinkage of H1.DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone H1 on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the H1.     

Vps75, a new yeast member of the NAP histone chaperone family

Homologues of nucleosome assembly protein 1 (NAP1) are found throughout eukaryotes. Here we identify and characterize a new NAP family histone chaperone from budding yeast, named Vps75. Purified Vps75 preferentially binds histone H3/H4 tetramers and is capable of assembling nucleosomes in vitro. In vivo, Vps75 is associated with the chromatin of both active and inactive genes and telomeres. Others have previously reported that Vps75 forms a complex with Rtt109, required for acetylation of histone H3 lysine 56 (H3 Lys-56). Cells lacking RTT109 are sensitive to hydroxyurea, pointing to a role in replication. We show that VPS75 is not required for H3 Lys-56 acetylation and that vps75Delta cells are insensitive to hydroxyurea, suggesting that although Rtt109 and Vps75 associate and are likely to be functionally connected, they also have separate roles.     

Immunological evidence for the existence of H1-like histone in yeast

In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt. Perchloric-acid-extracted proteins from whole cells contain only two fractions which comigrate with 'authentic' yeast high-mobility-group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross-reaction with anti-(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1. The isolation of 'authentic' yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti-HMG antiserum whereas the richer preparation also gives considerable cross-reaction with the anti-H1 antiserum. Immunoblotting analysis performed on the salt-extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti-H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.     

The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters

Eukaryotic gene expression starts off from a largely obstructive chromatin substrate that has to be rendered accessible by regulated mechanisms of chromatin remodeling. The yeast PHO5 promoter is a well known example for the contribution of positioned nucleosomes to gene repression and for extensive chromatin remodeling in the course of gene induction. Recently, the mechanism of this remodeling process was shown to lead to the disassembly of promoter nucleosomes and the eviction of the constituent histones in trans. This finding called for a histone acceptor in trans and thus made histone chaperones likely to be involved in this process. In this study we have shown that the histone chaperone Asf1 increases the rate of histone eviction at the PHO5 promoter. In the absence of Asf1 histone eviction is delayed, but the final outcome of the chromatin transition is not affected. The same is true for the coregulated PHO8 promoter where induction also leads to histone eviction and where the rate of histone loss is reduced in asf1 strains as well, although less severely. Importantly, the final extent of chromatin remodeling is not affected. We have also presented evidence that Asf1 and the SWI/SNF chromatin remodeling complex work in distinct parallel but functionally overlapping pathways, i.e. they both contribute toward the same outcome without being mutually strictly dependent.     

Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1

The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.     

sNASP, a histone H1-specific eukaryotic chaperone dimer that facilitates chromatin assembly

NASP has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but is widely distributed throughout eukaryotes both in its somatic and testicular forms. The secondary structure of the human somatic version consists mainly of clusters of α-helices and exists as a homodimer in solution. The protein binds nonspecifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1. The formation of the NASP-H1 complexes is mediated by the N-and C-terminal domains of histone H1 and does not involve the winged helix domain that is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.