We first identified SET2 in a selection for genes involved in the basal repression of GAL4. Three set2 mutants were isolated, the strongest of which was designated set2-1, which changes a highly conserved cysteine residue (C82) in the catalytic domain of Set2 to tyrosine. This suggests that Set2 represses basal transcription of GAL4 through its methyltransferase activity.
We identified other residues in the catalytic domain that are necessary for GAL4
repression (Fig. ). Conserved cysteine residues found in the SACI and SACII domains and highly conserved residues located in the SET domain are important for Set2 function. The structures of several SET domain proteins have been determined recently (7
), and two of the solved structures, S. pombe
Clr4 and Neurospora crassa
Dim-5, contain cysteine-rich SAC domains. The structures show that cysteines in the N-terminal SAC domain have a structural role in coordinating the binding of a triangular zinc cluster, while the cysteine corresponding to C201 in Set2 has been suggested to contact the C-terminal cysteine-rich SAC domain to form a cofactor-substrate binding site (22
). Given this structural information, we believe that the C82Y mutant alters the zinc cluster structure and that the enzyme loses activity because of structural changes. On the other hand, the C201A mutation should cause minimal structural changes but the purified protein may be unable to form an intact cofactor or substrate binding site in vitro. It is possible that such a binding site can be restored to some degree in vivo in the presence of other proteins. These structure-based interpretations are consistent with our results shown in Fig. . In addition, three of the mutations recovered in the selection, C97, H199, and Q112 (similar to Clr4 R320), were found to ablate HMT activity in previously characterized HMT enzymes, leading us to believe that the defects in set2
affect catalysis rather than protein-protein interactions (24
We found that the catalytic domain of Set2 has HMT activity in vitro (Fig. ) and, in agreement with a recent report (33
), that the prominent site of methylation is lysine 36 on histone H3. We showed that GST-Set2 cannot methylate a histone H3 substrate when lysine 36 is converted to an unmethylatable arginine, confirming its preference for lysine 36 (Fig. ).
We believe that the HMT activity of Set2 on H3 lysine 36 is responsible for its basal repression of GAL4 for four reasons. First, the set2-1 mutant (C82Y) isolated in our original screening, as well as the C201A mutant, is catalytically inactive in vitro (Fig. ). Second, these catalytically inactive mutants have a significantly reduced ability to repress the ΔUAS gal4::cat reporter gene (see Results and Fig. ). Third, the ability of Set2 to repress GAL4 expression is dependent on the availability of lysine 36 on H3 for methylation, because the hht2 K36R change causes a loss of repression of the ΔUAS gal4::cat reporter gene that is the same whether the SET2 gene is present or has been deleted (Fig. ). Finally, the chromatin immunoprecipitation experiments show that SET2 is directly responsible for methylation of lysine 36 at the GAL4 gene (Fig. ). The combination of genetic and biochemical evidence strongly suggests that repression of GAL4 by Set2 is mediated by methylation of lysine 36 on histone H3.
The repressive effects of Set2 methylation on transcription in vivo are in agreement with a previous report (33
). In that report, LexA-Set2 was found to repress transcription 20-fold when tethered to a CYC1-lexAop-lacZ
reporter. The differences in the level of repression by Set2 at GAL4
may be due to differences in the recruitment of Set2 to these promoters. In agreement with our results, a C201A mutation in LexA-Set2 resulted in a 50% loss in repression (33
). This partial effect could be due to the different activity levels of the mutant protein in vitro (where it was completely defective; Fig. ) versus that seen in vivo. Or perhaps Set2 has a repression function independent of its methylation activity.
It is not easy to reconcile our results regarding the repression of basal transcription of GAL4
with the numerous recent reports that Set2 binds to the elongating form of RNA polymerase II (17
). Perhaps Set2 acts as a backup system for transcriptional repression. Under conditions of repression, transcriptional repressors bind to the promoter region of regulated genes, preventing the assembly and subsequent clearance of an RNA polymerase II transcription complex. But occasionally, “leaky” transcription can occur under repressive conditions. Perhaps Set2 methylates the promoter and the coding part of the gene when this leaky transcription occurs, thus marking the chromatin and preventing subsequent transcription. It is also possible that the methylation of lysine 36 by Set2 has different functions at promoters than at coding regions of genes.
The actual repression mechanism resulting from lysine 36 methylation is still not known. One model is that the methylation of lysine 36 causes an alteration of nucleosome structure that is repressive in nature. To test this hypothesis, we conducted MNase protection assays on hht2
and HHT2 SET2
strains at the GAL4
promoter. We found no difference in digestion patterns, suggesting that nucleosome positioning had not been altered in the absence of methylation (data not shown). It is still possible that K36 methylation changes chromatin structure in a way that cannot be detected with MNase assays. A second model is that methylation of lysine 36 might recruit a chromodomain-containing protein that acts as a repressor of transcription. This would be similar to the mechanism used for the establishment of heterochromatin by HP1 binding to methyl lysine 9 on H3 (2
In summary, we have shown that Set2 methylation is involved in the repression of basal transcription of GAL4
. The fate of the methylated histones under conditions of transcriptional activation is unknown. It is possible that methylation of K36 at GAL4
is permanent and that its repressive effects are overcome through the recruitment of transcriptional activators. It is also possible that a demethylating enzyme exists. Finally, there may be a mechanism whereby methylated histones are replaced by unmethylated ones upon transcriptional activation (1