A conserved patch near the C terminus of histone H4 is required for genome stability in budding yeast

A screen of Saccharomyces cerevisiae histone alanine substitution mutants revealed that mutations in any of three adjacent residues, L97, Y98, or G99, near the C terminus of H4 led to a unique phenotype. The mutants grew slowly, became polyploid or aneuploid rapidly, and also lost chromosomes at a high rate, most likely because their kinetochores were not assembled properly. There was lower histone occupancy, not only in the centromeric region, but also throughout the genome for the H4 mutants. The mutants displayed genetic interactions with the genes encoding two different histone chaperones, Rtt106 and CAF-I. Affinity purification of Rtt106 and CAF-I from yeast showed that much more H4 and H3 were bound to these histone chaperones in the case of the H4 mutants than in the wild type. However, in vitro binding experiments showed that the H4 mutant proteins bound somewhat more weakly to Rtt106 than did wild-type H4. These data suggest that the H4 mutant proteins, along with H3, accumulate on Rtt106 and CAF-I in vivo because they cannot be deposited efficiently on DNA or passed on to the next step in the histone deposition pathway, and this contributes to the observed genome instability and growth defects.     

The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly

Two very similar H3 histones-differing at only four amino acid positions-are produced in Drosophila cells. Here we describe a mechanism of chromatin regulation whereby the variant H3.3 is deposited at particular loci, including active rDNA arrays. While the major H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition. In contrast to replication-coupled (RC) deposition, RI deposition does not require the N-terminal tail. H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast. RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification. Inheritance of newly deposited nucleosomes may then mark sites as active loci.     

Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.     

Global reorganization of budding yeast chromosome conformation in different physiological conditions

The organization of the genome is nonrandom and important for correct function. Specifically, the nuclear envelope plays a critical role in gene regulation. It generally constitutes a repressive environment, but several genes, including the GAL locus in budding yeast, are recruited to the nuclear periphery on activation. Here, we combine imaging and computational modeling to ask how the association of a single gene locus with the nuclear envelope influences the surrounding chromosome architecture. Systematic analysis of an entire yeast chromosome establishes that peripheral recruitment of the GAL locus is part of a large-scale rearrangement that shifts many chromosomal regions closer to the nuclear envelope. This process is likely caused by the presence of several independent anchoring points. To identify novel factors required for peripheral anchoring, we performed a genome-wide screen and demonstrated that the histone acetyltransferase SAGA and the activity of histone deacetylases are needed for this extensive gene recruitment to the nuclear periphery.     

Histone H3 localizes to the centromeric DNA in budding yeast

During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA.     

The histone fold domain of Cse4 is sufficient for CEN targeting and propagation of active centromeres in budding yeast

Centromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly. Cse4 is the CenH3 protein of the budding yeast Saccharomyces cerevisiae. Like all CenH3 proteins, Cse4 consists of a conserved histone fold domain (HFD) and a divergent N terminus (NT). The Cse4 NT contains an essential domain designated END (for essential N-terminal domain); deletion of END is lethal. To investigate the role of the Cse4 NT in centromere targeting, a series of deletion alleles (cse4DeltaNT) were analyzed. No part of the Cse4 NT was required to target mutant proteins to centromere DNA in the presence of functional Cse4. A Cse4 degron strain was used to examine targeting of a Cse4DeltaNT protein in the absence of wild-type Cse4. The END was not required for centromere targeting under these conditions, confirming that the HFD confers specificity of Cse4 centromere targeting. Surprisingly, overexpression of the HFD bypassed the requirement for the END altogether, and viable S. cerevisiae strains in which the cells express only the Cse4 HFD and six adjacent N-terminal amino acids (Cse4Delta129) were constructed. Despite the complete absence of the NT, mitotic chromosome loss in the cse4Delta129 strain increased only 6-fold compared to a 15-fold increase in strains overexpressing wild-type Cse4. Thus, when overexpressed, the Cse4 HFD is sufficient for centromere function in S. cerevisiae, and no posttranslational modification or interaction of the NT with other kinetochore component(s) is essential for accurate chromosome segregation in budding yeast.     

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.