Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants was generated in Saccharomyces cerevisiae that probes the contribution of each residue to nucleosome function. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.

It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of Sgo1p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint.During mitosis, chromatin goes through significant compaction and condensation to form metaphase chromosomes for segregation. While there is a wealth of information on the crucial roles played by chromatin structures and histone modifications in controlling transcription, replication, repair, and recombination (30), much less is known about how individual histones contribute mechanistically to mitotic progression and regulation.Forward and reverse genetic studies have suggested that histones, rather than being merely a part of the cargo during mitotic segregation, may play key roles in cell cycle progression and regulation. A histone H4 allele, hhf1-20, compromises the interaction between H4 and the centromere-specific H3 variant Cse4p, thus impeding centromeric functions and mitosis at the restrictive temperature (50). Two alleles of histone H2A (44) cause cold-sensitive growth defects and a significant increase in ploidy. This hyperploidy phenotype can be suppressed by mutations affecting a histone deacetylase, HDA1 (19). Similarly, the Gcn5p histone acetyltransferase genetically interacts with several inner kinetochore components and is physically mapped to the centromeric regions (57). Deleting the flexible tail domain of H3 and H4 results in mitotic delay (36) via a mechanism that can be suppressed by inhibiting the spindle assembly checkpoint activity (J.L. and M.H.K., unpublished data). Together, these data warrant a more thorough examination of how chromatin may proactively regulate the process of mitotic segregation.The center stage for mitotic segregation and control is the kinetochore, a large proteinaceous complex assembled on centromeres. The ultimate function of the kinetochore is to capture the spindle microtubules during mitosis. The kinetochore-spindle attachment ...