Histone H3 and H4 gene deletions in Saccharomyces cerevisiae

The genome of haploid Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 genes. Strains with deletions of each of these loci were constructed by gene replacement techniques. Mutants containing deletions of either gene set were viable, however meiotic segregants lacking both histone H3 and H4 gene loci were inviable. In haploid cells no phenotypic expression of the histone gene deletions was observed; deletion mutants had wild-type growth rates, were not temperature sensitive for growth, and mated normally. However, diploids homozygous for the H3-H4 gene deletions were slightly defective in their growth and cell cycle progression. The generation times of the diploid mutants were longer than wild-type cells, the size distributions of cells from exponentially growing cultures were skewed towards larger cell volumes, and the G1 period of the mutant cells was longer than that of the wild-type diploid. The homozygous deletion of the copy-II set of H3-H4 genes in diploids also increased the frequency of mitotic chromosome loss as measured using a circular plasmid minichromosome assay.     

Divergent Residues Within Histone H3 Dictate a Unique Chromatin Structure in Saccharomyces cerevisiae

Histones are among the most conserved proteins known, but organismal differences do exist. In this study, we examined the contribution that divergent amino acids within histone H3 make to cell growth and chromatin structure in Saccharomyces cerevisiae. We show that, while amino acids that define histone H3.3 are dispensable for yeast growth, substitution of residues within the histone H3 α3 helix with human counterparts results in a severe growth defect. Mutations within this domain also result in altered nucleosome positioning, both in vivo and in vitro, which is accompanied by increased preference for nucleosome-favoring sequences. These results suggest that divergent amino acids within the histone H3 α3 helix play organismal roles in defining chromatin structure.     

Histone H2A is required for normal centromere function in Saccharomyces cerevisiae

Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G(2)-M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere-kinetochore function during chromosome segregation.     

Transcription of multiple cell wall protein-encoding genes in Saccharomyces cerevisiae is differentially regulated during the cell cycle

The yeast cell wall consists of an internal skeletal layer and an outside protein layer. The synthesis of both β-1,3-glucan and chitin, which together form the cell wall skeleton, is cell cycle-regulated. We show here that the expression of five cell wall protein-encoding genes (CWP1, CWP2, SED1, TIP1 and TIR1) is also cell cycle-regulated. TIP1 is expressed in G1 phase, CWP1, CWP2 and TIR1 are expressed in S/G2 phase, and SED1 in M phase. The data suggest that these proteins fulfil distinct functions in the cell wall.     

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

The human β-like globin genes are arranged as a clusterof five genes (ε, Gγ, Aγ, δ and β) in the order of theirtemporal expression. The human embryonic ε-globin geneis expressed in the blood island of the embryonic yolk sacand is silenced completel     

Bub2 is a cell cycle regulated phospho-protein controlled by multiple checkpoints

During mitotic exit, a small GTPase Tem1 needs to be activated. During most of the cell cycle, Tem1 activity is antagonized by a GTPase activating complex (GAP) composed of Bub2 and Bfa1. Bfa1 protein has cell cycle regulated phosphorylation depending upon the Polo-like kinase Cdc5. This phosphorylation dissociates Bfa1 from Tem1 and thus relieves the inhibition of Tem1 by the GAP complex. Bub2 and Bfa1 are also required to prevent mitotic exit when there is DNA damage, spindle damage or spindle misorientation at G(2)/M phase. While Cdc5 inhibits Bfa1/Bub2, mutating the Cdc5 phosphorylation sites on Bfa1 does not have a strong activating effect on Bub2/Bfa1, suggesting there must be additional regulation in this pathway. Here we report that Bub2 protein also has cell cycle regulated phosphorylation. This phosphorylation is partially dependent upon the Polo-like kinase Cdc5 and is consistent with negative regulation of the Bub2/Bfa1 GAP complex. Spindle damage or spindle misorientation prevents Bub2 phosphorylation. The spindle damage effect is dependent upon the spindle assembly checkpoint components Mad2 and Mps1. Thus like Bfa1, Bub2 protein is also controlled both during mitotic exit and in response to cell cycle checkpoints. Bub2 phosphorylation is likely to be controlled by a novel kinase.     

Regulation of gene expression during meiosis in Saccharomyces cerevisiae: SPR3 is controlled by both ABFI and a new sporulation control element.

The SPR3 gene encodes a sporulation-specific homolog of the yeast Cdc3/10/11/12 family of bud neck filament proteins. It is expressed specifically during meiosis and sporulation in Saccharomyces cerevisiae. Analysis of the sporulation-specific regulation of SPR3 has shown that it is strongly activated under sporulating conditions but shows low levels of expression under nonsporulating conditions. A palindromic sequence located near the TATA box is essential to the developmental regulation of this gene and is the only element directly activating SPR3 at the right time during sporulation. Within the palindrome is a 9-bp sequence, gNCRCAAA(A/T) (midsporulation element [MSE]), found in the known control regions of three other sporulation genes. A previously identified ABFI element is also needed for activation. The MSE has been shown to activate a heterologous promoter (CYC1) in a sporulation-specific manner. Related sequences, including an association of MSE and ABFI elements, have been found upstream of other genes activated during the middle stage of S. cerevisiae sporulation. One group of these may be involved in spore coat formation or maturation.     

Histone H4 lysine 20 of Saccharomyces cerevisiae is monomethylated and functions in subtelomeric silencing

Histones undergo post-translational modifications that are linked to important biological processes. Previous studies have indicated that lysine methylation correlating with closed or repressive chromatin is absent in the budding yeast Saccharomyces cerevisiae, including at H4 lysine 20 (K20). Here we provide functional evidence for H4 K20 monomethylation (K20me1) in budding yeast. H4 K20me1 is detectable on endogenous H4 by western analysis using methyl-specific antibodies, and the signal is abrogated by H4 K20 substitutions and by competition with H4 K20me1 peptides. Using chromatin immunoprecipitation, we show that H4 K20me1 levels are highest at heterochromatic locations, including subtelomeres, the silent mating type locus, and rDNA repeats, and lowest at centromeres within euchromatin. Further, an H4 K20A substitution strongly reduced heterochromatic reporter silencing at telomeres and the silent mating type locus and led to an increase in subtelomeric endogenous gene expression. The correlation between the location of H4 K20me1 and the effect of the H4 K20A substitution suggests that this modification plays a repressive function. Our findings reveal the first negative regulatory histone methylation in budding yeast and indicate that H4 K20me1 is evolutionarily conserved from simple to complex eukaryotes.