A novel domain in Set2 mediates RNAPII interaction.
Although Set2 is known to bind to the phosphorylated CTD of RNAPII, the region(s) within Set2 responsible for this association is poorly defined (19
). Thus, we generated a series of Set2 mutant yeast expression constructs that contained a C-terminal Flag epitope tag (Fig. ) and used them in co-IP studies with antibodies generated against RNAPII. Either full-length SET2
, vector-only control, or the indicated SET2
mutant was expressed in a set2
deletion mutant (set2Δ
), and WCEs were prepared. As expected, immunoprecipitation of full-length Set2-Flag resulted in coprecipitation of RNAPII as detected by immunoblotting with anti-phospho-CTD antibodies (Fig. ). We previously reported that specific deletion of the WW domain in Set2 does not disrupt Set2-RNAPII coprecipitation, and so we hypothesized that the coiled-coil region of Set2 may be responsible (42
). Unexpectedly, we found that a precise deletion of the coiled-coil region in Set2 (amino acids 548 to 618) did not disrupt this interaction (data not shown), indicating that a previously undefined region in Set2 mediates this association. As shown in Fig. , we found through further Set2 truncations that a region at the C terminus of Set2, encompassing amino acid residues 619 to 733, is both necessary and sufficient to mediate the interaction of Set2 with RNAPII (compare last two lanes). We therefore termed this region the SRI domain.
FIG. 1. Identification of a novel region in Set2 required for RNAPII binding. (A) Schematic representation of the Set2 constructs used to probe for RNAPII interaction. The SET domain along with the AWS domain, postSET domain (PS), WW domain, and coiled-coil motif (more ...)
We next sought to determine the precise boundaries of the SRI domain. To accomplish this, we generated additional Set2 constructs containing N- and C-terminal truncations of the SRI region (Fig. ) and used them in co-IP analyses as described for Fig. . Results revealed that N-terminal truncation of the SRI domain beyond Set2 amino acid 619 abolished RNAPII binding. However, binding was still possible with a C-terminal truncation up to amino acid 718 of Set2 (Fig. ), thereby identifying the boundaries of the SRI domain as amino acids 619 to 718.
Due to the possibility that the observed interaction between the SRI domain and RNAPII shown in Fig. might have been influenced by the high levels of recombinant Set2 protein produced (as these constructs are expressed from a plasmid using the highly active ADH1 promoter), we genomically tagged Set2 at amino acid 733 or 618 with a triple Flag sequence and reexamined its association with RNAPII. As shown in Fig. , full-length Set2 (Set2-3Flag) again coimmunoprecipitated RNAPII as analyzed by immunoblot analysis with the anti-phospho-CTD antibodies. In contrast, a form of Set2 with the SRI domain deleted [Set2(1-618)-3Flag] resulted in the abolition of RNAPII interaction. We also confirmed these results by examining the protein associations of Set2 by affinity purification, which revealed that the readily detectable subunits of RNAPII (Rpb1 and Rpb2) were only observed in purifications involving full-length Set2 (Fig. ). Additionally, while Rpb1 and Rpb2 were detected by mass spectrometry in gel excised bands from the full-length Set2 purification, these proteins were not detected in a parallel gel region excised from the form of Set2 with the SRI domain deleted (data not shown). Collectively, these data confirm the importance of the SRI domain in mediating the Set2-RNAPII interaction.
FIG. 2. The SRI domain is required for interaction of Set2 with RNAPII. (A) Yeast strains containing full-length Set2 (Set2-3Flag) or a form of Set2 without the SRI domain [Set2(1-618)-3Flag] were made via genomic tagging with the 3xFlag epitope. WCEs of these (more ...) The SRI domain of Set2 is conserved and interacts with the phosphorylated CTD in vitro.
Previous studies have suggested that Set2 association with RNAPII is dependent, in part, on the Paf1 transcription elongation complex (12
). Thus, it was a formal possibility that the SRI domain linked Set2 to the polymerase by indirect protein association. To test whether the SRI domain of Set2 is responsible for direct association with the CTD of RNAPII, we generated a variety of MBP fusions of Set2 and examined their ability to associate with a recombinant GST-CTD fusion protein that was either unmodified (GST-yCTD) or phosphorylated by CTDK-I (GST-yPCTD). Using a reverse far Western approach (see reference 31
), the GST-CTD fusions were resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with MBP fusion proteins carrying full-length Set2 or only the SRI domain of Set2 [Set2(619-733)
]. Results revealed that both the full-length form of Set2 and the SRI domain of Set2 preferentially bound to the phosphorylated CTD (Fig. ). To independently confirm this interaction and further address whether other regions of Set2 may bind to the phosphorylated CTD in vitro, we transferred increasing amounts of MBP fusions of Set2 lacking the SRI or containing only the SRI domain [Set2(1-618)
, respectively] to nitrocellulose and probed with a CTDK-I-phosphorylated GST-[32
P]CTD fusion. As shown in Fig. , this far Western approach revealed that while the SRI domain of Set2 bound efficiently to the GST-[32
P]CTD fusion, Set2 lacking the SRI domain did not. While Fig. shows a 3.5-h exposure, it is noteworthy that a 90-h exposure revealed a potential weak interaction of Set2(1-618)
to phosphorylated CTD (data not shown); however, it is unclear whether such interaction is physiologically relevant (Fig. to ) (31
). In summary, our results show that the SRI domain in Set2 binds directly to the phospho-CTD of RNAPII. Thus, the ability of the Paf1 complex to modulate Set2 activity is likely an indirect consequence of the fact that this complex can regulate CTD phosphorylation (28
FIG. 3. The SRI domain of Set2 binds synergistically to doubly modified CTD repeats. (A) Reverse far Western analysis. GST-yCTD and CTDK-I-phosphorylated GST-CTD (GST-yPCTD) fusion proteins were subjected to SDS-PAGE and transferred to nitrocellulose. Membranes (more ...)
FIG. 4. Deletion of the SRI domain in Set2 abolishes H3 K36 dimethylation. (A) Yeast nuclear extracts prepared from set2Δ cells or the indicated genomically tagged strains in the BY4742 background were probed with antibodies against dimethylated lysine (more ...)
Next, we examined the specificity of the SRI domain for binding phospho-epitopes by BIACORE analysis with three-repeat synthetic CTD peptides that were phosphorylated in each repeat at either Ser2 (2-phospho), Ser5 (5-phospho), or both (2 + 5-phospho). As a control, a Ser-phosphorylated peptide that mimics the charge state of the 2 + 5-phospho peptide (6PC) was included. Sensor chips containing these CTD peptides were reacted with the SRI domain of Set2 [Set2(619-733)], and binding was monitored. Surprisingly, we found that the Set2 SRI domain bound preferentially to CTD repeats that were doubly phosphorylated (Fig. , compare 2 + 5-phospho curve with 2-phospho and 5-phospho curves). Moreover, because these response curves were obtained after subtracting the contribution of the nonspecific control peptide (6PC), this binding depends on the presence of both Ser2P and Ser5P in the context of the CTD heptad repeat sequence. Based on additional BIACORE experiments involving titrated amounts of the Set2 SRI domain (not shown), we determined that the Set2 SRI domain binds to the 2 + 5-phospho peptide (relative to the control) with an apparent dissociation constant of about 6 μM. It is important to note that the ability of the SRI domain to bind to the nonspecific control peptide was nearly equivalent to that found for binding to the individually phosphorylated peptides (data not shown). We take this result to suggest that the SRI domain of Set2 has a specific requirement for Ser2- and Ser5-phosphorylated CTD epitopes. Collectively, these results reveal a novel and selective requirement for a specific CTD phosphorylation pattern in Set2 binding to RNAPII.
Given these findings, we next asked whether the SRI domain of Set2 is a conserved phospho-CTD-binding motif found in other proteins in budding yeast and beyond. By performing a PSI-BLAST search, we determined that the SRI domain of Set2 was unique to this enzyme alone in budding yeast (data not shown). However, the SRI domain showed significant homology to the C-terminal regions of proteins in other species that also displayed domain organizations similar to that of Set2 (AWS, SET, postSET, and WW), suggesting that these proteins may be the functional homologs of budding yeast Set2 and function with RNAPII (Table ). Interestingly, the proteins identified in Table represent only a subset of proteins that the SMART database revealed to contain AWS, SET, and postSET domains (>70), suggesting that not every putative histone methyltransferase that contains an AWS, SET, and postSET is by default a functional homolog of Set2. Indeed, recent evidence shows that the Drosophila melanogaster
Ash1 protein, which falls into the Set2 family of HMTs (by way of having an AWS domain rather than an archetypal PreSET domain), is an H3 lysine 4 methyltransferase (4
). These results suggest that the SRI domain is a probable indicator of RNAPII-interacting enzymes that catalyze K36 methylation. To determine whether any of the putative SRI domains we identified by our PSI-BLAST search would actively bind to the phospho-CTD, we expressed and purified from bacteria a GST fusion protein carrying the C-terminal 178 residues of the human Huntington interacting protein B (HYPB) that includes the region of homology to Set2's SRI domain (Table ). Using the far Western approach as described for Fig. , we found that similar to Set2, the SRI-containing region in HYPB interacts efficiently with a CTDK-I-phosphorylated GST-[32
P]CTD fusion (data not shown). Furthermore, additional BIACORE analyses (as described for Fig. ) revealed that the human SRI domain displays binding properties nearly identical to those of the budding yeast domain (H. P. Phatnani, A. L. Greenleaf, and P. Zhou, unpublished results). Taken together, these results suggest that the SRI domain is a highly conserved and novel phospho-CTD-interacting domain.
Putative Set2 homologs identified by PSI-BLAST searching with the SRI domain
The SRI-RNAPII interaction is required for H3 K36 methylation.
Given the conserved nature and potential importance of the SRI domain to Set2's cellular function, we next investigated the consequences of deleting this domain. Although studies suggest that the CTD and its proper phosphorylation are necessary for K36 methylation, it has not been formally excluded that the phospho-CTD might regulate the enzymatic activity of Set2 (19
). To determine if the loss of the SRI domain would result in a loss of genome-wide K36 methylation, we measured the K36 dimethylation levels in strains containing WT Set2 or Set2 with the SRI domain genomically deleted. Nuclei were prepared from these strains and then resolved on an SDS-PAGE gel, followed by Western blotting with an antibody specific to dimethylated K36. Results revealed that deletion of the SRI domain in Set2 abolishes global H3-K36 dimethylation (Fig. ). As a control, we examined the levels of H3 in parallel gels with an antibody specific to the C terminus of H3, which revealed that the levels of histones were similar in both nuclei preparations. Importantly, the nuclei of both strains showed the presence of Set2 by Western blot analysis using an anti-Flag antibody (Fig. ). This result indicates that the deletion of the SRI domain does not influence the nuclear localization of Set2 or significantly affect its stability. The requirement of the SRI domain for K36 methylation was independently confirmed in parallel studies in which a different strain background (W303) was genomically tagged either at the C terminus or at the beginning of the SRI domain at residue 618 (data not shown). In addition, we used the ChIP assay to analyze chromatin modifications at a gene-specific resolution and also observed a loss of K36 methylation at the SCC2
gene when the SRI domain of Set2 is removed (Fig. ). Analysis of the PMA1
, and ADH1
genes yielded similar results (data not shown).
To test the possibility that the SRI domain itself might regulate Set2's catalytic activity, we analyzed recombinant full-length Set2 or SRI-deleted MBP-Set2 fusion proteins in HMT assays with chicken oligo-nucleosomes. Results showed that both forms of the enzyme were equally active for K36 methylation, indicating that the SRI domain is not required for the catalytic activity of Set2 in vitro (Fig. ). In fact, we found that a region of Set2 encompassing the AWS, SET, and postSET domains (amino acid residues 1 to 261 in Set2) is fully active for histone methylation in vitro, indicating that the C terminus of Set2 does not intrinsically regulate its HMT activity (data not shown).
Set2 methylation influences transcription elongation and RNAPII occupancy on genes.
Growth phenotypes observed in the presence of the drug 6AU are frequently used as indicators of defects in transcription elongation (8
). We therefore asked whether deletion of SET2
, or prevention of the Set2-RNAPII association by deletion of the SRI domain, would exhibit 6AU-dependent phenotypes. We began by examining the 6AU phenotypes caused by Set2 deletion in several strain backgrounds. Various WT (W303, BY4742/SC288C, and YCB652) and matched set2Δ
strains were grown on control medium (no drug) or medium containing 6AU. The parent strain YCB652 contained the integrated URA3
gene, which is required for the 6AU assay, while others were transformed with the URA3
plasmid pRS316 (38
). The survival and colony sizes of each strain were monitored after several days of growth and compared to those on control plates. As shown in Fig. , we found that deletion of SET2
in these strain backgrounds resulted in a significant resistance phenotype to 6AU. Similar results were also observed when we used medium containing mycophenolic acid, another drug that reveals elongation defects but through a mechanism unique from that of 6AU (data not shown). Furthermore, we analyzed a dst1
null strain of the BY4742 background and observed the characteristic 6AU sensitivity known to exist for this mutant (Fig. ) (3
FIG. 5. Deletion of SET2 results in an elongation phenotype and an alteration of RNAPII occupancy on genes. (A) Various strains containing either WT, set2Δ, or dst1Δ alleles were plated on synthetic dextrose-uracil medium with or without 6AU (100 (more ...)
Given that previous studies have demonstrated that a proper response to 6AU is the induced expression of the IMD2
gene, which is a result of the elongation machinery's response to depleted nucleotide pools (35
), we sought to verify that the resistance phenotype observed in the set2Δ
deletion mutant was not due to an aberrant effect on the cellular metabolism of 6AU. Using semiquantitative RT-PCR, we monitored the expression levels of IMD2
(an RNAPIII-transcribed gene used as a control) in the presence or absence of 6AU. As shown in Fig. , we found that the expression of the IMD2
gene was increased to equal degrees in both WT and set2Δ
strains in the presence of the drug, confirming that the loss of Set2 results in a bona fide transcription elongation defect. Importantly, the IMD2
gene was not induced in the absence of 6AU for either the WT or set2Δ
strains, indicating that Set2 does not act to repress the basal expression of this gene (Fig. ). In addition to our results with Set2, 6AU resistance has also been observed from the deletion or mutation of a variety of other elongation factors, including Chd1, Bye1, Isw1, and forkhead factor 1 (2
Since transcription elongation defects are typically correlated with changes in the occupancy and distribution of RNAPII along genes, we therefore asked whether the loss of Set2 would result in an alteration in RNAPII levels on actively transcribed genes. Using an antibody that recognizes the general levels of RNAPII irrespective of its phosphorylation status (34
), we examined RNAPII levels on the promoters and coding regions of active genes in WT and set2Δ
strains by ChIP. As shown in Fig. , we found that RNAPII levels in the set2
deletion mutant were significantly increased in the middle to late coding region of the actively transcribing SCC2
gene compared to the WT control strain. Interestingly, the gene locations that showed an RNAPII increase were also the same locations determined to be highly methylated by Set2 (Fig. ), suggesting the possibility that a relationship may exist between regions of chromatin highly methylated at K36 and RNAPII occupancy potential. We next examined a variety of other active genes in the set2
deletion mutant to determine how general this RNAPII defect would be. We examined the promoter and coding regions of TOM1
, and FIR1
for the presence of RNAPII as described above and found a similar pattern of RNAPII increase in the set2
deletion mutant as was observed for SCC2
(data not shown). We addressed the possibility that the observed increases in RNAPII might be a result of a general increase in transcript formation for these genes in the absence of Set2. Indeed, Set2 has been shown to play a role in the basal repression of GAL4
). We therefore examined the expression of the genes indicated above by semiquantitative RT-PCR and observed that the increased density of RNAPII did not correlate with any change in the steady-state mRNA levels (Fig. and data not shown). These mRNA results were also confirmed independently by examining the gene expression microarray profiles found in WT and set2Δ
cells (N. Krogan and J. Greenblatt, personal communication). Furthermore, we also examined TBP levels by ChIP at the promoters of several genes listed above (SCC2
) and found no significant increases in TBP occupancy in the set2
deletion mutant (data not shown). Our data indicate that Set2 does not function as a basal repressor of the genes analyzed, but rather it affects the precise levels of RNAPII on genes, further supporting the 6AU results suggesting that Set2 can influence RNAPII elongation.
The above results suggest Set2 is important for transcription elongation, but they do not reveal whether this function of Set2 is dependent on its association with RNAPII and/or K36 methylation. To test if loss of the interaction between Set2 and RNAPII is responsible for the elongation defect, we assayed the growth of strains with SRI deleted [Set2(1-618)-3Flag], SET2 deleted, and the Set2-3Flag strains by using 6AU. We observed that deletion of the SRI domain resulted in a resistance to 6AU that was similar to that of the set2 deletion mutant, indicating that the interaction between Set2 and RNAPII is necessary for the normal transcription elongation functions of Set2 (Fig. ).
FIG. 6. K36 methylation influences transcription elongation. (A) Genomically tagged strains containing either full-length Set2 (Set2-3Flag) or Set2 with the SRI domain deleted [Set2(1-618)-3Flag] were generated and assayed, as in Fig. , for growth (more ...)
We next asked whether K36 methylation per se is important for the activity of Set2 in this process. In one case, we transformed set2Δ
cells with a plasmid coding for either full-length Set2 (SET2
) or a form of Set2 containing a point mutation (set2R195G
) that has been shown to abolish K36 methylation activity in vitro and in vivo (39
). We found that expression of SET2
in the set2Δ
strain nearly restored WT levels of 6AU sensitivity (Fig. ). However, set2Δ
cells expressing set2R195G
showed resistance to the drug (Fig. ), consistent with a role for K36 methylation in the elongation process. In the second case, we asked whether amino acid substitutions at K36 that prevent methylation (K36A and K36R) would result in resistance to 6AU and RNAPII density increases. As shown in Fig. , the K36A and K36R strains were significantly resistant to 6AU compared to the WT H3 strain, whereas strains with mutations at other sites of methylation (K4 and K79) or sites of phosphorylation (serine 10) were not. In addition, the 6AU resistance caused by the K36A or K36R mutations was not suppressed by mutation of lysine 4 (Fig. , K4R/K36R). Significantly, we also found the same pattern of increased RNAPII density for the SCC2
gene in the K36A strain as with the set2Δ
strain (Fig. ), suggesting that the specific lack of K36 methylation is the primary cause of the 6AU phenotype and RNAPII defect. These data strongly implicate the methylation by Set2 as being functionally important in the elongation process.