Histone H3 methylation on Lys36 was recently attributed to the SET domain-containing protein Set2 in S. cerevisiae
). In an effort to further characterize this protein, we used single-step transformation to place a TAP tag on the C terminus of Set2. Following purification and analyses by SDS-PAGE and mass spectrometry, we discovered that a substoichiometric amount of RNA polymerase II (RNAPII) copurified with Set2.
This physical interaction suggested that methylation of histone H3 Lys36 might be intimately linked to transcription. ChIP analyses demonstrated that Set2 localizes primarily to the coding regions of three genes that were tested, and this correlated well with the locations of histone H3 Lys36 methylation. Consistent with the notion that the recruitment of Set2 is linked to elongation by RNAPII, the presence of Set2 was found to correlate with active transcription when ChIP was performed on the GAL1
gene following induction with galactose. Strahl et al. tethered Set2 to a heterologous promoter and demonstrated that, under these conditions, Set2 represses transcription (54
). However, we found little or no Set2 or histone H3 Lys36 methylation in various promoter regions, suggesting that Set2 activity is not normally directed towards this region. Deletion of the SET2
gene caused slight sensitivity to 6-AU and a large reduction in β-galactosidase produced by a reporter gene. Both the sensitivity to 6-AU and the reduced β-galactosidase synthesis from the reporter gene that we used in our experiments have been used as indicators that a gene is involved in enhancing elongation by RNAPII (3
). A similar suggestion has been made based on the enhanced sensitivity to 6-AU generated when a SET2
deletion is combined with a deletion of DST1
, which encodes TFIIS (28
). Follow-up ChIP experiments showed that the concentration of RNAPII is lower across the lacZ
gene in a set2
Δ background than in a wild-type strain. Therefore, we propose that wild-type Set2 normally has a positive role in transcription. This experiment did not, however, prove that Set2 specifically stimulates elongation by RNAPII.
Phosphorylation and dephosphorylation of the CTD of the largest subunit of RNAPII, Rpb1, are required for gene regulation (22
). Phosphorylation on Ser5 of the CTD heptapeptide repeats, YSPTSPS, by the Kin28 subunit of the general transcription factor TFIIH is associated with early transcriptional elongation, whereas Ser2 phosphorylation by Ctk1 is found at later elongation steps (10
). Our Western blot analyses demonstrated that both types of RNAPII copurify with Set2, again implying that the recruitment of Set2 to a transcription unit and histone H3 methylation on Lys36 might be cotranscriptional. It is striking that the distribution of Set2 along various genes, strong in the coding region and weak in the promoter-proximal and 3′ untranslated regions, is virtually identical to the distribution of Ser2-phosphorylated CTD (10
) and the Ser2 kinase, Ctk1 (M. Kim, S. H. Ahn, and S. Buratowski, unpublished data). Moreover, deletion of CTK1
results in substantially less recruitment of Set2 to the PMA1
gene and virtually eliminates histone H3 Lys36 methylation, indicating that Lys36 methylation depends on Ser2 phosphorylation of the CTD during transcriptional elongation.
The WW domains of Ess1/Pin1 and Rsp5 are known to interact with the proline-rich CTD repeats of RNAPII (8
). Since our ChIP experiments showed that deletion of the C-terminal region of Set2, including its WW domain, reduced the recruitment of Set2 and essentially eliminated its methylation activity, we propose that there is a physical interaction between the WW domain of Set2 and the Ser2-phosphorylated CTD of RNAPII that stabilizes the association of Set2 with RNAPII and triggers the histone H3 methylation activity of Set2. Consistent with this conclusion, Li et al. recently also found that Set2 copurifies with RNAPII and showed that Set2 can bind to high concentrations of a Ser2-phosphorylated CTD peptide (29
). Although this study found that deletion of the WW domain of Set2 eliminates the association between RNAPII and Set2, we have observed that Set2 (Δ476-733)-TAP, which lacks the WW domain, still coimmunoprecipitates with some RNAPII (N. J. Krogan and J. Greenblatt, unpublished data) and is still recruited, albeit less efficiently, to actively transcribed genes (Fig. ). Xiao et al. (68
) have found that a C-terminal portion of Set2 that is distal to its WW domain is sufficient to bind RNAPII. Therefore, a site on RNAPII other than the Ser2-phosphorylated CTD is also likely to mediate an interaction with Set2.
To further pursue Set2's role in transcriptional elongation, the presence of histone H3 Lys36 methylation and the recruitment of Set2 were examined on the PMA1
gene when genes encoding subunits of the Paf1 complex were deleted. The Paf1 complex contains five subunits (Paf1, Cdc73, Rtf1, Leo1, and Ctr9) and has recently been implicated, both biochemically and genetically, in the process of transcriptional elongation (11
). Specifically, deletions of genes encoding components of the Paf1 complex cause sensitivity to 6-AU and genetically interact with mutations in genes encoding other known elongation factors. Furthermore, the Paf1 complex interacts physically with both RNAPII and Spt16/Pob3, the yeast homologue of the human elongation factor FACT (39
). ChIP experiments have also shown that the Paf1 complex colocalizes with RNAPII in the coding regions of various genes (25
) and, like Set2, declines in the region beyond the poly(A) signal (Kim et al., unpublished). We have shown here that when genes encoding two components of the Paf1 elongation complex, Cdc73 and Rtf1, are deleted, Set2 recruitment is significantly reduced and histone H3 methylation is virtually eliminated.
Consistent with a functional connection between Set2 and the Paf1 complex, synthetic growth defects were observed when a set2
deletion was individually combined with deletions of genes encoding all five subunits of the Paf1 complex. The SET2
gene also interacts with the genes encoding two other putative elongation factors, Soh1 and Chd1. Mutations in SOH1
were originally identified as suppressors of HPR1
), a component of the transcription elongation complex TREX (56
). We have recently found that a soh1
deletion also interacts genetically with genes encoding a number of other known elongation factors, including TFIIS
, and causes significant sensitivity to 6-AU and a marked decrease in expression of the lacZ
gene on the plasmid p416GAL1
(Krogan and Greenblatt, unpublished). The CHD1
gene interacts genetically with a number of genes encoding elongation factors (11
; Krogan and Greenblatt, unpublished) and was recently implicated in termination by RNAPII (1
). Moreover, we recently found that Chd1 is associated with casein kinase II and Spt16/Pob3 (25
) and can be effectively cross-linked to the coding regions of a number of genes (Kim et al., unpublished). The dependence of Set2 function on the Paf1 complex and the CTD kinase Ctk1, as well as the many genetic interactions between the SET2
gene and genes encoding known positive elongation factors, strengthens our conclusion that Set2 function is cotranscriptional and that Set2 may act as an activator of elongation.
Surprisingly, synthetic growth defects were obtained when a set2
deletion was combined with deletions of genes encoding all seven components of the Set3 complex (41
). The Set3 complex has a negative effect on the expression of certain meiosis-specific genes, perhaps because the Set3 complex contains two histone deacetylases, Hos2 and Hst1, as well as Set3. The synthetic phenotypes generated when a set2
deletion is combined with deletions of genes encoding components of the Set3 complex suggest, however, that the Set3 complex may also have a positive role in transcription by RNAPII. This idea is supported by the recent observation that at least two components of the Set3 complex, Set3 and, surprisingly, Hos2, are recruited to actively transcribed genes (65
deletion also generated synthetic growth defects when combined with deletions of genes encoding components of the Set1 complex, COMPASS. COMPASS methylates Lys4 of histone H3, a modification that has been shown to be needed for effective silencing at telomeres and ribosomal DNA loci (7
). Interestingly, components of COMPASS as well as histone H3 Lys4 methylation, like Set2 and components of the Paf1 complex, localize to the transcribed regions of various genes and interact genetically with a number of known or suspected elongation factors (5
; our unpublished data). Moreover, methylation of Lys4 by COMPASS, like methylation of Lys36 by Set2, also requires components of the Paf1 complex, which associates with COMPASS (26
). Genetic interactions among components of three different Set protein-containing complexes imply that these complexes are functionally redundant and may all associate directly or indirectly with RNAPII and function during transcriptional elongation.
Our data and other published studies on COMPASS and Set2 have uncovered a remarkable coordination of RNAPII phosphorylation with histone H3 methylation during transcriptional elongation, as illustrated in Fig. . COMPASS is recruited specifically to the early transcribed region (26
), and this depends on both the Paf1 complex and phosphorylation of the RNAPII CTD on Ser5 by Kin28. The consequence of COMPASS recruitment is histone H3 trimethylation on Lys4 in the same region (37
). Upon further elongation by RNAPII, Ser5 phosphorylation declines and is replaced by Ser2 phosphorylation mediated by Ctk1 (10
). We found that Set2 is recruited to this region, where it methylates Lys36 of histone H3, and the recruitment of Set2 depends on both the Paf1 complex and phosphorylation of RNAPII by Ctk1 on Ser2, as has also been observed by Li et al. (28
) and Xiao et al. (68
). Finally, in the region beyond the poly(A) signal, Set2 disappears and histone H3 Lys36 declines in concert with dephosphorylation of RNAPII on Ser2 (10
) and the disappearance of the Paf1 complex (Kim et al., unpublished).
Model for the coupling of histone methylation to transcriptional elongation and the phosphorylation of RNAPII in S. cerevisiae. See the text for details.
Δ and set2
Δ double mutants also have synthetic growth defects. Bre1, an E3 ubiquitin ligase, is associated with Rad6 and Lge1, and all three are required for ubiquitination of histone H2B, a modification which is necessary for methylation of histone H3 on Lys4 by COMPASS (13
; our unpublished data). Therefore, disruption of Lys4 methylation by deleting genes encoding components of COMPASS or the Bre1/Lge1 complex results in a growth defect when SET2
is also deleted. This further confirms the functional redundancy between the Lys36 and Lys4 methylations on histone H3. Interestingly, SET2
also genetically interacts with HTZ1
, which encodes a variant histone, H2A (20
). This genetic interaction may predict a role for Htz1 in histone ubiquitination, histone methylation, and/or transcriptional elongation.