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.     

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.     

Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS

COMPASS, the yeast homolog of the mammalian MLL complex, is a histone H3 lysine 4 (H3K4) methylase consisting of Set1 (KMT2) and seven other polypeptides, including Cps35, the only essential subunit. Histone H2B monoubiquitination by Rad6/Bre1 is required for both H3K4 methylation by COMPASS, and H3K79 methylation by Dot1. However, the molecular mechanism for such histone crosstalk is poorly understood. Here, we demonstrate that histone H2B monoubiquitination controls the binding of Cps35 with COMPASS complex. Cps 35 is required for COMPASS' catalytic activity in vivo, and the addition of exogenous purified Cps35 to COMPASS purified from a Deltarad6 background results in the generation of a methylation competent COMPASS. Cps35 associates with the chromatin of COMPASS-regulated genes in a H2BK123 monoubiquitination-dependent but Set1-independent manner. Cps35 is also required for proper H3K79 trimethylation. These findings offer insight into the molecular role of Cps35 in translating the H2B monoubiquitination signal into H3 methylation.     

Trimethylation of Histone H3 Lysine 36 by Human Methyltransferase PRDM9 Protein

PRDM9 (PR domain-containing protein 9) is a meiosis-specific protein that trimethylates H3K4 and controls the activation of recombination hot spots. It is an essential enzyme in the progression of early meiotic prophase. Disruption of the PRDM9 gene results in sterility in mice. In human, several PRDM9 SNPs have been implicated in sterility as well. Here we report on kinetic studies of H3K4 methylation by PRDM9 in vitro indicating that PRDM9 is a highly active histone methyltransferase catalyzing mono-, di-, and trimethylation of the H3K4 mark. Screening for other potential histone marks, we identified H3K36 as a second histone residue that could also be mono-, di-, and trimethylated by PRDM9 as efficiently as H3K4. Overexpression of PRDM9 in HEK293 cells also resulted in a significant increase in trimethylated H3K36 and H3K4 further confirming our in vitro observations. Our findings indicate that PRDM9 may play critical roles through H3K36 trimethylation in cells.     

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.     

Di-methyl H4 lysine 20 targets the checkpoint protein Crb2 to sites of DNA damage

Histone lysine methylation is an important chromatin modification that can be catalyzed to a mono-, di-, or tri-methyl state. An ongoing challenge is to decipher how these different methyllysine histone marks can mediate distinct aspects of chromatin function. The fission yeast checkpoint protein Crb2 is rapidly targeted to sites of DNA damage after genomic insult, and this recruitment requires methylation of histone H4 lysine 20 (H4K20). Here we show that the tandem tudor domains of Crb2 preferentially bind the di-methylated H4K20 residue. Loss of this interaction by disrupting either the tudor-binding motif or the H4K20 methylating enzyme Set9/Kmt5 ablates Crb2 localization to double-strand breaks and impairs checkpoint function. Further we show that dimethylation, but not tri-methylation, of H4K20 is required for Crb2 localization, checkpoint function, and cell survival after DNA damage. These results argue that the di-methyl H4K20 modification serves as a binding target that directs Crb2 to sites of genomic lesions and defines an important genome integrity pathway mediated by a specific methyl-lysine histone mark.