A role for histone H2B during repair of UV-induced DNA damage in Saccharomyces cerevisiae

To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Delta and rad52Delta mutants but not in rad6Delta or rad18Delta mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Delta) or error-free (rad30Delta) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Delta mutation. When combined with a ubc13Delta mutation, which is also epistatic with rad5Delta, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.     

Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair

The modification of newly synthesized histones H3 and H4 by type B histone acetyltransferases has been proposed to play a role in the process of chromatin assembly. The type B histone acetyltransferase Hat1p and specific lysine residues in the histone H3 NH(2)-terminal tail (primarily lysine 14) are redundantly required for telomeric silencing. As many gene products, including other factors involved in chromatin assembly, have been found to participate in both telomeric silencing and DNA damage repair, we tested whether mutations in HAT1 and the histone H3 tail were also sensitive to DNA-damaging agents. Indeed, mutations both in specific lysine residues in the histone H3 tail and in HAT1 resulted in sensitivity to methyl methanesulfonate. The DNA damage sensitivity of the histone H3 and HAT1 mutants was specific for DNA double-strand breaks, as these mutants were sensitive to the induction of an exogenous restriction endonuclease, EcoRI, but not to UV irradiation. While histone H3 mutations had minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in the recombinational repair of DNA double-strand breaks. Epistasis analysis indicates that the histone H3 and HAT1 mutants may influence DNA double-strand break repair through Asf1p-dependent chromatin assembly.     

Tandem bromodomains in the chromatin remodeler RSC recognize acetylated histone H3 Lys14

The coordination of chromatin remodeling with chromatin modification is a central topic in gene regulation. The yeast chromatin remodeling complex RSC bears multiple bromodomains, motifs for acetyl-lysine and histone tail interaction. Here, we identify and characterize Rsc4 and show that it bears tandem essential bromodomains. Conditional rsc4 bromodomain mutations were isolated, and were lethal in combination with gcn5Delta, whereas combinations with esa1 grew well. Replacements involving Lys14 of histone H3 (the main target of Gcn5), but not other H3 or H4 lysine residues, also conferred severe growth defects to rsc4 mutant strains. Importantly, wild-type Rsc4 bound an H3 tail peptide acetylated at Lys14, whereas a bromodomain mutant derivative did not. Loss of particular histone deacetylases suppressed rsc4 bromodomain mutations, suggesting that Rsc4 promotes gene activation. Furthermore, rsc4 mutants displayed defects in the activation of genes involved in nicotinic acid biosynthesis, cell wall integrity, and other pathways. Taken together, Rsc4 bears essential tandem bromodomains that rely on H3 Lys14 acetylation to assist RSC complex for gene activation.     

Point mutations in the yeast histone H4 gene prevent silencing of the silent mating type locus HML.

The N-terminal serine and four conserved lysine residues near the N-terminus of yeast histone H4 are acetylated. We found that a mutation that changed the fourth lysine to alanine resulted in specific derepression of the silent mating type locus HML, while mutations that altered the N-terminal serine or the first three lysines had only minor phenotypic effects. Our results support an active role for histone H4 in the silencing of gene expression at this locus.     

A lesson learned from the H3.3K27M mutation found in pediatric glioma

Glioblastoma (GBM) is the most aggressive primary brain tumor in human. Recent studies on high-grade pediatric GBM have identified two recurrent mutations (K27M and G34R/V) in genes encoding histone H3 (H3F3A for H3.3 and HIST1H3B for H3.1).^1,2 The two histone H3 mutations are mutually exclusive and give rise to tumors in different brain compartments.³ Recently, we⁴ and others⁵ have shown that the histone H3 K27M mutation specifically altered the di- and tri-methylation of endogenous histone H3 at Lys27. Genome-wide studies using ChIP-seq on H3.3K27M patient samples indicate a global reduction of H3K27me3 on chromatin. Remarkably, we also found a dramatic enrichment of H3K27me3 and EZH2 (the catalytic subunit H3K27 methyltransferase) at hundreds of gene loci in H3.3K27M patient cells. Here, we discuss potential mechanisms whereby H3K27me3 is enriched at chromatin loci in cells expressing the H3.3K27M mutation and report effects of Lys-to-Met mutations of other well-studied lysine residues of histone H3.1/H3.3 and H4 on the corresponding endogenous lysine methylation. We suggest that mutation(s) on histones may be found in a variety of human diseases, and the expression of mutant histones may help to address the function of histone lysine methylation and possibly other modifications in mammalian cells.     

Methylation of histone H3 mediates the association of the NuA3 histone acetyltransferase with chromatin

The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3Delta phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.     

Global assessment of combinatorial post-translational modification of core histones in yeast using contemporary mass spectrometry. LYS4 trimethylation correlates with degree of acetylation on the same H3 tail

A global view of all core histones in yeast is provided by tandem mass spectrometry of intact histones H2A, H2B, H4, and H3. This allowed detailed characterization of >50 distinct histone forms and their semiquantitative assessment in the deletion mutants gcn5Delta, spt7Delta, ahc1Delta, and rtg2Delta, affecting the chromatin remodeling complexes SAGA, SLIK, and ADA. The "top down" mass spectrometry approach detected dramatic decreases in acetylation on H3 and H2B in gcn5Delta cells versus wild type. For H3 in wild type cells, tandem mass spectrometry revealed a direct correlation between increases of Lys(4) trimethylation and the 0, 1, 2, and 3 acetylation states of histone H3. The results show a wide swing from 10 to 80% Lys(4) trimethylation levels on those H3 tails harboring 0 or 3 acetylations, respectively. Reciprocity between these chromatin marks was apparent, since gcn5Delta cells showed a 30% decrease in trimethylation levels on Lys(4) in addition to a decrease of acetylation levels on H3 in bulk chromatin. Deletion of Set1, the Lys(4) methyltransferase, was associated with the linked disappearance of both Lys(4) methylation and Lys(14) and Lys(18) or Lys(23) acetylation on H3. In sum, we have defined the "basis set" of histone forms present in yeast chromatin using a current mass spectrometric approach that both quickly profiles global changes and directly probes the connectivity of modifications on the same histone.