Histone deacetylase 3 is required for centromeric H3K4 deacetylation and sister chromatid cohesion

We describe here the role of histone deacetylase 3 (HDAC3) in sister chromatid cohesion and the deacetylation of histone H3 Lys 4 (H3K4) at the centromere. HDAC3 knockdown induced spindle assembly checkpoint activation and sister chromatid dissociation. The depletion of Polo-like kinase 1 (Plk1) or Aurora B restored cohesion in HDAC3-depleted cells. HDAC3 was also required for Shugoshin localization at centromeres. Finally, we show that HDAC3 depletion results in the acetylation of centromeric H3K4, correlated with a loss of dimethylation at the same position. These findings provide a functional link between sister chromatid cohesion and the mitotic "histone code".     

A charge-based interaction between histone H4 and Dot1 is required for H3K79 methylation and telomere silencing: identification of a new trans-histone pathway

Saccharomyces cerevisiae cells lacking Dot1 exhibit a complete loss of H3K79 methylation and defects in heterochromatin-mediated silencing. To further understand the mechanism of Dot1-mediated methylation, the substrate requirement of Dot1 was determined. This analysis found that Dot1 requires histone H4 for in vitro methyltransferase activity and the histone H4 tail for Dot1-mediated methylation in yeast. Mutational analyses demonstrated that the basic patch residues (R(17)H(18)R(19)) of the histone H4 N-terminal tail are required for Dot1 methyltransferase activity in vitro as well as Dot1-mediated histone H3K79 methylation in vivo. In vitro binding assays show that Dot1 can interact with the H4 N-terminal tail via the basic patch residues. Furthermore, an acidic patch at the C terminus of Dot1 is required for histone H4 tail binding in vitro, histone H3K79 di- and trimethylation in vivo, and proper telomere silencing. Our data suggest a novel trans-histone regulatory pathway whereby charged residues of one histone are required for the modification of another histone. These findings not only provide key insights into the mechanism of Dot1 histone methylation but also illustrate how chromatin-modifying enzymes engage their nucleosomal substrates in vivo.     

CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae

We previously purified a yeast protein kinase that specifically hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II largest subunit and showed that this CTD kinase consists of three subunits of 58, 38, and 32 kDa. We have now cloned, sequenced, and characterized CTK1, the gene encoding the 58 kDa alpha subunit. The CTK1 gene product contains a central domain homologous to catalytic subunits of other protein kinases, notably yeast CDC28, suggesting that the 58 kDa subunit is catalytic. Cells that carry a disrupted version of the CTK1 gene lack the characterized CTD kinase activity, grow slowly and are cold-sensitive, demonstrating that the CTK1 gene product is essential for CTD kinase activity and normal growth. While ctk1 mutant cells do contain phosphorylated forms of the RNA polymerase II largest subunit, these forms differ from those found in wild type cells, implicating CTK1 as a component of the physiologically significant CTD phosphorylating machinery. As befitting an enzyme with a nuclear function, the N-terminal region of the CTK1 protein contains a nuclear targeting signal.     

Role of SGS1 and SLX4 in maintaining rDNA structure in Saccharomyces cerevisiae

To address the role of SGS1 in controlling genome stability we previously identified several slx mutants that require SGS1 for viability. Here, we report the isolation and characterization of temperature-sensitive (ts) SGS1 alleles in cells lacking SLX4. At the non-permissive temperature (37 degrees C) sgs1-ts slx4 cells progress through S-phase and arrest growth as large-budded cells with at least a 2C DNA content. Analysis of the integrity of the replicated DNA by pulsed-field gel electrophoresis revealed that chromosome XII (ChrXII) was uniquely altered, as it was unable to enter the gel. This defect was specific to the tandem rDNA repeats on ChrXII and occurred as cells progressed through S-phase at 37 degrees C. Reciprocal-shift experiments revealed that viability and ChrXII migration can be restored by allowing Sgs1 to act between G2/M and the subsequent G1 phase. These results suggest that Sgs1 and Slx4 are not required for bulk DNA synthesis but play redundant roles in maintaining rDNA structure during DNA replication.     

Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4

Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD_4–6) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4''s nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD_4–6 reduce PHD_4–6''s binding ability and MLL4''s catalytic activity. PHD_4–6''s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s''s interference with PHD_4–6''s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation.     

An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae

P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA‐repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4‐mediated genotoxicity of a well‐known genotoxin, aflatoxin B1 (AFB₁). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac, a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB₁, the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB₁, and illustrate the usefulness of the CYP3A4‐expressing, DNA‐repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity. Environ. Mol. Mutagen. 58:217–227, 2017. © 2017 Wiley Periodicals, Inc.     

SAS-mediated acetylation of histone H4 Lys 16 is required for H2A.Z incorporation at subtelomeric regions in Saccharomyces cerevisiae

The yeast SAS (Something About Silencing) complex and the histone variant H2A.Z have both previously been linked to an antisilencing function at the subtelomeric regions. SAS is an H4 Lys 16-specific histone acetyltransferase complex. Here we demonstrate that the H4 Lys 16 acetylation by SAS is required for efficient H2A.Z incorporation near telomeres. The presence of H4 Lys 16 acetylation and H2A.Z synergistically prevent the ectopic propagation of heterochromatin. Overall, our data suggest a novel antisilencing mechanism near telomeres.     

DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 °C), but is not affected at the permissive temperature (23 °C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.     

An α-specific gene,SAG1 is required for sexual agglutination in Saccharomyces cerevisiae

Summary Seven -specific mutants specifically defective in sexual agglutinability were isolated. The other mating functions exhibited by these mutants, designated sag mutants, such as the production of pheromone and response to a mating pheromone, were normal. While the MAT sag1 cells did not agglutinate with wild-type a cells, the MAT sag1 cells did, indicating that the SAG1 gene is expressed only in cells. The mutations were semi-dominant and fell into a single complementation group, SAG1, which was mapped near met3 on chromosome X. Complementation analysis showed that sag1 and aga1, the latter being a previously reported -specific mutation, were mutations in the same gene.     

Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association

The amino-terminal histone tails are subject to covalent post-translational modifications such as acetylation, methylation, and phosphorylation. In the histone code hypothesis, these exposed and unstructured histone tails are accessible to a repertoire of regulatory factors that specifically recognize the various modified histones, thereby generating altered chromatin structures that mediate specific biological responses. Here, we report that lysine (Lys) 79 of histone H3, which resides in the globular domain, is methylated in eukaryotic organisms. In the yeast Saccharomyces cerevisiae, Lys 79 of histone H3 is methylated by Dot1, a protein shown previously to play a role in telomeric silencing. Mutations of Lys 79 of histone H3 and mutations that abolish the catalytic activity of Dot1 impair telomeric silencing, suggesting that Dot1 mediates telomeric silencing largely through methylation of Lys 79. This defect in telomeric silencing might reflect an interaction between Sir proteins and Lys 79, because dot1 and Lys 79 mutations weaken the interaction of Sir2 and Sir3 with the telomeric region in vivo. Our results indicate that histone modifications in the core globular domain have important biological functions.     

The DNA methylation locus DDM1 is required for maintenance of gene silencing in Arabidopsis

To investigate the relationship between cytosine methylation and gene silencing in Arabidopsis, we constructed strains containing the ddm1 hypomethylation mutation and a methylated and silenced PAI2 tryptophan biosynthetic gene (MePAI2) that results in a blue fluorescent plant phenotype. The ddm1 mutation had both an immediate and a progressive effect on PAI gene silencing. In the first generation, homozygous ddm1 MePAI2 plants displayed a weakly fluorescent phenotype, in contrast to the strongly fluorescent phenotype of the DDM1 MePAI2 parent. After two generations of inbreeding by self-pollination, the ddm1/ddm1 lines became nonfluorescent. The progressive loss of fluorescence correlated with a progressive loss of methylation from the PAI2 gene. These results indicate that methylation is necessary for maintenance of PAI gene silencing and that intermediate levels of DNA methylation are associated with intermediate gene silencing. The results also support our earlier hypothesis that ddm1 homozygotes act as “epigenetic mutators” by accumulating heritable changes in DNA methylation that can lead to changes in gene expression.