Because Set1/COMPASS has proven to be challenging to characterize structurally, given its size and subunit complexity, we generated a homology model of the Set1 catalytic domain (Fig. ) to gain a better insight into the molecular mechanism of H3K4 methylation by Set1/COMPASS. This model allowed us to identify residues within the active site of Set1 that may contribute to its product specificity or the degree of lysine methylation. Utilizing this model, we identified several active-site residues that may participate in substrate binding or catalysis. One such residue is Tyr1052 within the lysine binding channel (Fig. ).
FIG. 1. Homology model of the yeast Set1 catalytic domain. (a) Ribbon representation of the secondary structure of the Set1 catalytic domain illustrating the SET domain (blue), the inserted SET motif (iSET, green), and the PostSET domain (red). A histone H3 substrate (more ...)
To assess the potential role of Tyr1052 in Set1's methyltransferase activity and product specificity, we developed yeast strains in which Set1 can be mutated and incorporated into COMPASS for both in vivo and in vitro studies (Fig. ). Using this system, we mutated the Phe/Tyr switch residue Tyr1052 to phenylalanine in Set1 (Fig. ). The Y1052F mutant yields stable COMPASS, as demonstrated by the stability of Set1 (Fig. ). Set1 carrying the Y1052F mutation exhibits greater H3K4 trimethyltransferase activity than the wild-type complex in vivo, in agreement with the Phe/Tyr switch model (Fig. ). An analysis of H3K4 methylation in the Tyr1052 mutant strain reveals an increase in the ratio of H3K4me3 to H3K4me2 relative to their levels in the wild type (Fig. ).
FIG. 2. H3K4 methyltransferase activity of the Set1 Y1052F mutant in vivo. (A) Schematic representation of the yeast construct used for the mutational analyses of the SET domain of Set1. Yeast cells with the genomic SET1 gene replaced with the HIS3 marker are (more ...)
This phenomenon is also true for the in vitro methyltransferase activity of COMPASS. Purified COMPASS either from the wild-type strain or from the strain harboring a Set1 Y1052F mutant demonstrates similar stability within Set1 (Fig. ). However, complexes carrying Set1 Y1052F are much more active in H3K4 trimethylation than the wild-type enzyme (Fig. ). Similar to the results of the in vivo studies (Fig. ), the ratio of H3K4me3 to H3K4me2 is also increased in the in vitro studies, in contrast to wild-type COMPASS in which H3K4me2 is the most-abundant species. Furthermore, our analysis of the molecular composition of the wild-type and the Y1052F-mutated COMPASS indicated that the subunit composition of the complex is not altered by this single point mutation within Set1's catalytic pocket (Fig. ).
FIG. 3. Intrinsically enhanced in vitro histone H3K4 methyltransferase activity of Set1(Y1052F) proteins within COMPASS. (A) The levels of Set1 and TAP-tagged Cps60 proteins in purified COMPASS were determined. Increasing concentrations of TAP-tagged Cps60-purified (more ...)
In previous studies, we demonstrated that Cps40 (Spp1) and Cps60 (Bre2) can regulate the pattern of H3K4 trimethylation and postulated that this may occur through direct regulation of the catalytic pocket of Set1 (24
). Since we have identified a single point mutation within the catalytic pocket of Set1(Y1052F) that can enhance the H3K4 trimethylation activity of the enzyme, we wanted to determine whether this mutation suppresses the trimethylation loss in a cps40
) null strain. To this end, cps40
) and set1
double null cells were transformed with either the vector, the vector containing wild-type Set1, or the vector containing the Y1052F mutant. The Set1 levels were similar in the wild-type and mutant Set1-transformed cells, as assayed by Western analyses of whole-cell extracts (Fig. ) and purified COMPASS (Fig. ). In contrast, the H3K4 trimethylation levels are greatly increased in the Y1052F mutant relative to the levels with wild-type Set1, with no observable change in the H3K4 dimethylation level, demonstrating that the cps40
deletion is suppressed by Y1052F in vivo (Fig. ). This observation indicates that Cps40 (Spp1)'s function is to regulate the H3K4 trimethylation activity of Set1/COMPASS through this residue. Similarly, COMPASS purified from a cps40
) null strain is defective in H3K4 trimethylation; however, the Y1052F mutation within Set1 suppresses this deficiency in an in vitro histone methyltransferase assay (Fig. ). This observation is further supported by a previously published study indicating that Cps40 (Spp1) and Set1 can interact directly with each other in yeast (5a
FIG. 4. Complementation of H3K4 trimethylation in a cps40 (spp1)-deficient COMPASS both in vivo and in vitro. (A) Wild-type and Y1052F mutant Set1 protein levels in a cps40 null strain. Anti-Set1 and anti-H14 Western analyses were performed as described for Fig. (more ...)
The studies presented here demonstrate that Tyr1052 of Set1 is inhibitory to H3K4 trimethylation, as wild-type COMPASS null for Cps40 (Spp1) is incapable of properly trimethylating H3K4 both in vivo and in vitro (Fig. ). However, the presence of Cps40 (Spp1) or the mutation of Tyr1052 to phenylalanine that results in the deletion of the hydroxyl group from the phenol side chain promotes H3K4 trimethylation by Set1 (Fig. ). Since our previous studies demonstrated that both Cps40 (Spp1) and Cps60 (Bre2) are required for proper H3K4 trimethylation, we also tested the role of Y1052F mutation in H3K4 trimethylation in the absence of Cps60 (Bre2). Indeed, we find that the mutation of Tyr1052 to phenylalanine promotes H3K4 trimethylation by Set1 in the absence of Cps60 (Bre2) (Fig. ). Interestingly, H3K4 dimethylation in a cps60
) null strain is also complemented by a Y1052F mutation of Set1 (Fig. ). Monoubiquitination of histone H2B by Rad6/Bre1 is required for proper H3K4 methylation by COMPASS (7
). We therefore tested whether the Set1 Y1052F mutant can compensate for the loss of Rad6. As a control for our in vivo studies, we tested for the Set1 levels in these cells and have confirmed that the deletion of rad6
or the point mutation of the Set1 catalytic domain did not alter Set1's stability in cells (Fig. ). As shown by the results in Fig. , the in vivo methylation activity of COMPASS in a rad6
Δ background is not suppressed by the Set1 Y1052F mutation. This is also true for the in vitro studies (Fig. ). When similar levels of Set1 within COMPASS purified from either the wild-type or rad6
null background were tested for H3K4 methylation, Y1052F did not suppress the loss of Rad6's effect on H3K4 methylation (Fig. ).
FIG. 5. Y1052F Set1 suppresses H3K4 di- and trimethylation defects in a cps60 (bre2) null strain, while H3K4 methylation loss caused by the loss of Rad6 is refractory to this mutation. (A) Wild-type and Y1052F mutant Set1 protein expression in a cps60 (bre2) (more ...)
We previously demonstrated that H2B monoubiquitination/H3K4 methylation cross talk is regulated via the Cps35 (Swd2) subunit of COMPASS, as this subunit interacts with the chromatin of COMPASS-regulated genes in an H2B monoubiquitination-dependent manner and is required for proper H3K4 methylation (14
). Furthermore, the Cps35 (Swd2) ortholog in mammals, Wdr82, also interacts with chromatin in a H2B monoubiquitination-dependent manner and is a specific component of the mammalian Set1/COMPASS and not of the MLL complexes (mammalian COMPASS-like complexes) (32
). Recently, it has been proposed that Cps35 (Swd2) is ubiquitinated by the Rad6/Bre1 complex when both Cps35 (Swd2) and ubiquitin are overexpressed in yeast cells (31
). Based on the observation that Cps40's association with chromatin is reduced in a strain carrying point mutations in the proposed ubiquitination sites of Cps35 (Swd2), it has been postulated that this histone cross talk in yeast is further regulated via recruitment of Cps40 (Spp1) through Cps35 (Swd2) (31
). However, our data presented in this report indicate that Cps40 (Spp1) regulates the pattern of H3K4 trimethylation independently of H2B and/or Cps35 (Swd2) monoubiquitination. Cps40 (Spp1) regulates H3K4 trimethylation through a Phe/Tyr switch within Set1's catalytic pocket (Fig. ), and the loss of H3K4 methylation as a result of the loss of H2B monoubiquitination is not suppressed by the Y1052F mutation (Fig. ). Therefore, it appears that these two pathways, Cps40 (Spp1)-dependent H3K4 methylation and monoubiquitination-dependent H3K4 methylation, are functioning via different mechanisms.
To further investigate the relative roles of Cps35 (Swd2) and Cps40 (Spp1) in histone cross talk, we compared the chromatin recruitment of each protein in the presence and absence of Rad6. ChIP was performed with myc-tagged Cps35 in wild-type and either rad6
Δ or H2B(K123R) strains. As previously shown, Cps35's association with chromatin is Rad6- and H2B monoubiquitination dependent (Fig. ). To test the proposed model (31
) that Cps40 (Spp1) interacts with chromatin in a Cps35 (Swd2)-dependent manner, we have utilized a cps35
deletion strain, recently generated by Nagy and colleagues (19
). Cps60 was TAP-tagged in cps35
Δ strains, and COMPASS was purified from this background. The resulting complex was analyzed by employing multidimensional protein identification technology (MudPIT). In this method, we use the spectral counts, i.e., the total number of tandem mass spectrum-matching peptides from a protein. Spectral counts have been defined as excellent markers of protein abundance in shotgun proteomics analyses (15
). Dividing the spectral count by molecular weight defines a spectral abundance factor (20
), and hence, proteins of different sizes can be compared. Previously, we demonstrated that wild-type COMPASS contains one copy of the Set1, Cps50 (Swd1), Cps40 (Spp1), and Cps30 (Swd3) subunits; less than one copy of Cps35 (Swd2); two copies of Cps60 (Bre2); and at least three copies of Cps25 (Sdc1). Since the loss of Cps35 (Swd2) results in some loss of COMPASS stability, we normalized the levels of each COMPASS subunit from both wild-type and cps35
) null backgrounds to the level of Set1. Based on our MudPIT analysis of COMPASS purified from a cps35
) null background, the level of Cps40 (Spp1) appears to be unchanged relative to the level of Set1 (Fig. ). We see an increase in the levels of Cps60 and Cps25 in COMPASS from the cps35
null background. This observation is to be expected, as we have tagged Cps60 in this background and Cps60 and Cps25 form a heteromeric complex. Since the Set1 level is reduced in a cps35
null background, we therefore observe an increase in the level of the Cps60/25 heteromeric complex. Overall, contrary to a previously published study (31
), our data indicate that the association of Cps40 (Spp1) with COMPASS does not require Cps35 (Swd2), as we are able to purify the same ratio of Cps40 (Spp1) with COMPASS in the absence of Cps35 (Swd2) as in its presence (Fig. ). Our data also indicate that the loss of H2B monoubiquitination does not alter the association of Cps40 with chromatin of COMPASS-regulated genes, contrary to a previously published study (31
) (Fig. ). We have also previously demonstrated that Cps60's association with COMPASS and COMPASS-regulated genes is unchanged in strains defective for H2B monoubiquitination (14
FIG. 6. Cps40 (Spp1) can interact with COMPASS independently of monoubiquitinated histone H2B and Cps35 (Swd2). (A and B) ChIP of the GAL1 gene through myc-tagged Cps35 in the presence and absence of ubiquitinated H2B. Myc-Cps35 ChIP from wild-type (WT) and (more ...)
Collectively, these studies indicate that while Cps35 (Swd2) is a key mediator of cross talk between H2B monoubiquitination and H3K4 methylation, as initially proposed (14
), Cps40's (Spp1's) association with COMPASS does not depend on the presence of Cps35 (Swd2) within the complex or its prior monoubiquitination. Thus, while both Cps35 and Cps40 regulate the transitioning of methyl states of H3K4 by COMPASS, each does so by a distinct mechanism, demonstrating the complexity of H3K4 methylation regulation by COMPASS.
In histone methyltransferases that are functional as single polypeptides, the Phe/Tyr switch controls product specificity by modulating the binding of the active-site water molecule (5
). In di- and trimethyltransferases, the presence of a phenylalanine or other hydrophobic residue in the Phe/Tyr switch site modulates the affinity for the active-site water, allowing multiple methylation reactions (s). In contrast, the methyltransferase activity and product specificity of Set1 are dependent on the subunits constituting COMPASS. Although SET1 would be predicted to be an H3K4 monomethyltransferase due to Phe/Tyr switch residue Tyr1052 (Fig. ), Cps40 (Spp1) and Cps60 (Bre2) have a predominant role in determining its product specificity in the context of COMPASS. From the results of this study, we propose that these subunits could associate directly with the catalytic domain of Set1, possibly through interactions with the histone substrate binding cleft or the PostSET motif (Fig. and ). Indeed, a recent structural study of the human MLL1 catalytic domain supports this model (27
). In the crystal structure of an MLL1 ternary complex, the histone H3 peptide is bound in a deep channel formed by the inserted SET motif (iSET) helix and the PostSET domain, as illustrated in our homology model of the Set1 catalytic domain (27
) (Fig. ). The shifting of the iSET region away from the histone binding cleft results in an active site that is relatively open and solvent exposed when compared to the structure of the H3K9-specific methyltransferase Dim-5 and is believed to contribute to the weak methyltransferase activity of the free enzyme. The activity of MLL1 is greatly stimulated in the presence of the RbBP5 and Ash2L core subunits, which have been proposed to facilitate the closure of the iSET helix over the active site. Correlatively, we have shown that the association of Cps40 and Cps60, the functional homologs of Ash2L in yeast, with Set1 promote H3K4 di- and trimethylation, presumably through an analogous mechanism (24
). Future studies are necessary to elucidate the nature of the interactions among Set1, Cps40, and Cps60, as well as the mechanism by which these interactions alter the activity and product specificity of COMPASS.
FIG. 7. Model illustrating the function of Cps40 in the regulation of histone methyltransferase activity of Set1/COMPASS. (A) Cps40 and Cps60 (yellow diamond) induce a conformational change of Set1/SET domain, pulling back Tyr1052 (light-purple trapezoid) so (more ...)