Histone H3-H4 Tetramer Research Articles

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.

Continuous Histone H2B and Transcription-Dependent Histone H3 Exchange in Yeast Cells outside of Replication

We investigated the dynamics of histone-DNA interactions in yeast by using inducible forms of epitope-tagged histones H2B and H3. Chromatin assembly of newly synthesized histones was assessed by chromatin immunoprecipitation in G1-arrested cells to prevent replication-coupled histone incorporation. We find that while histone deposition within a subtelomeric region is strictly linked to DNA replication, histone H2B is continuously incorporated at the promoter and coding regions of both transcriptionally active and inactive loci. In contrast, incorporation of histone H3 occurs only at active genes, being predominant at the promoter and showing a dynamics along the gene that inversely correlates with the average nucleosomal density. Similar results were obtained with N-terminally truncated H2B and H3 variants. We infer that replication-independent incorporation of H2B and H3 are distinct events, each occurring independently of the histone tail, and that nucleosome loss at active promoters reflects a dynamic equilibrium between histone deposition and dissociation.

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.

Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.

Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.

The identity of conformational states of reconstituted and native histone octamers.

Histone octamers were reconstituted from the following preparations: (a) natural histone H3-H4 tetramer and histone H2A-H2B dimer, either selectively extracted from chromatin with solutions chloride or prepared by dissociation of the natural octamer; (b) acid-denatured core histones, either an unfractionated mixture or individually purified proteins. Complexes assembled from these histones elute from exclusion chromatography columns with octamer size as verified by cross-linking with dimethylsuberimidate. The reconstituted octamers all crystallize in the same form of helical tubes as the natural octamer.

Nucleohistone Assembly: Sequential Binding of Histone H3-H4 Tetramer and Histone H2A-H2B Dimer to DNA

Nucleohistone Assembly: Sequential Binding of Histone H3-H4 Tetramer and Histone H2A-H2B Dimer to DNA