Catalytic and Functional Roles of Conserved Amino Acids in the SET Domain of the S. cerevisiae Lysine Methyltransferase Set1

In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.     

Dot1p modulates silencing in yeast by methylation of the nucleosome core

DOT1 was originally identified as a gene affecting telomeric silencing in S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates approximately 90% of histone H3. In dot1delta cells, silencing is compromised and silencing proteins become redistributed at the expense of normally silenced loci. We suggest that methylation of histone H3 lysine 79 limits silencing to discrete loci by preventing the binding of Sir proteins elsewhere along the genome. Because Dot1p and histone H3 are conserved, similar mechanisms are likely at work in other eukaryotes.     

Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.     

Global loss of Set1-mediated H3 Lys4 trimethylation is associated with silencing defects in Saccharomyces cerevisiae

Post-translational histone modifications, such as acetylation, phosphorylation, ubiquitination, and methylation, have been correlated with regulation of gene expression. In Saccharomyces cerevisiae, Set1 has been identified as the sole histone methyltransferase required for histone H3 lysine 4 (Lys(4)) methylation. Yeast cells that do not express Set1 have several apparent phenotypes, including slow growth and defects in telomere, HML, and rDNA silencing. However, the mechanism by which the Set1 methyltransferase mediates differential histone H3 methylation (mono-, di-, and tri-) is still not understood, and the involvement of domains or regions in Set1 contributing to H3 Lys(4) methylation has not been well characterized. In this study, the N terminus of Set1 was shown to be important for global and gene specific histone H3 trimethylation. We show that Set1 trimethyl-defective mutants can rescue a set1Delta slow growth defect. In contrast, Set1 trimethyl mutants were defective in telomere, rDNA, HML, and HMR silencing. Taken together, these data suggest that histone H3 Lys(4) trimethylation is required for proper silencing, while mono- and/or dimethylation is sufficient for cell growth.     

Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6

The DNA of eukaryotes is wrapped around nucleosomes and packaged into chromatin. Covalent modifications of the histone proteins that comprise the nucleosome alter chromatin structure and have major effects on gene expression. Methylation of lysine 4 of histone H3 by COMPASS is required for silencing of genes located near chromosome telomeres and within the rDNA (Krogan, N. J, Dover, J., Khorrami, S., Greenblatt, J. F., Schneider, J., Johnston, M., and Shilatifard, A. (2002) J. Biol. Chem. 277, 10753-10755; Briggs, S. D., Bryk, M., Strahl, B. D., Cheung, W. L., Davie, J. K., Dent, S. Y., Winston, F., and Allis, C. D. (2001) Genes. Dev. 15, 3286-3295). To learn about the mechanism of histone methylation, we surveyed the genome of the yeast Saccharomyces cerevisiae for genes necessary for this process. By analyzing approximately 4800 mutant strains, each deleted for a different non-essential gene, we discovered that the ubiquitin-conjugating enzyme Rad6 is required for methylation of lysine 4 of histone H3. Ubiquitination of histone H2B on lysine 123 is the signal for the methylation of histone H3, which leads to silencing of genes located near telomeres.     

Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase

We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development.     

Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase

Yeast disruptor of telomeric silencing-1 (DOT1) is involved in gene silencing and in the pachytene checkpoint during meiotic cell cycle. Here we show that the Dot1 protein possesses intrinsic histone methyltransferase (HMT) activity. When compared with Rmt1, another putative yeast HMT, Dot1 shows very distinct substrate specificity. While Rmt1 methylates histone H4, Dot1 targets histone H3. In contrast to Rmt1, which can only modify free histones, Dot1 activity is specific to nucleosomal substrates. This was also confirmed using native chromatin purified from yeast cells. We also demonstrate that, like its mammalian homolog PRMT1, Rmt1 specifically dimethylates an arginine residue at position 3 of histone H4 N-terminal tail. In surprising contrast, methylation by Dot1 occurs in the globular domain of nucleosomal histone H3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis suggests that H3 lysine 79 is trimethylated by Dot1. The intrinsic nucleosomal histone H3 methyltransferase activity of Dot1 is certainly a key aspect of its function in gene silencing at telomeres, most likely by directly modulating chromatin structure and Sir protein localization. In agreement with a role in regulating localization of histone deacetylase complexes like SIR, an increase of bulk histone acetylation is detected in dot1- cells.