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• W2051163721 abstract "•Set1 protein levels are upregulated in response to transcription and H3K4 methylation•Loss of feedback control disrupts normal H3K4 methylation patterns along genes•Loss of H3K4me2 and H3K4me3 in immunoblots of extracts masks gene-specific defects•H3K4me3 turns over faster at SAGA-dependent genes than at TFIID-dependent genes Methylation of histone H3 lysine 4 by the Set1 subunit of COMPASS correlates with active transcription. Here, we show that Set1 levels are regulated by protein degradation in response to multiple signals. Set1 levels are greatly reduced when COMPASS recruitment to genes, H3K4 methylation, or transcription is blocked. The degradation sequences map to N-terminal regions that overlap a previously identified autoinhibitory domain, as well as the catalytic domain. Truncation mutants of Set1 that cause under- or overexpression produce abnormal H3K4 methylation patterns on transcribed genes. Surprisingly, SAGA-dependent genes are more strongly affected than TFIID-dependent genes, reflecting differences in their chromatin dynamics. We propose that careful tuning of Set1 levels by regulated degradation is critical for the establishment and maintenance of proper H3K4 methylation patterns. Methylation of histone H3 lysine 4 by the Set1 subunit of COMPASS correlates with active transcription. Here, we show that Set1 levels are regulated by protein degradation in response to multiple signals. Set1 levels are greatly reduced when COMPASS recruitment to genes, H3K4 methylation, or transcription is blocked. The degradation sequences map to N-terminal regions that overlap a previously identified autoinhibitory domain, as well as the catalytic domain. Truncation mutants of Set1 that cause under- or overexpression produce abnormal H3K4 methylation patterns on transcribed genes. Surprisingly, SAGA-dependent genes are more strongly affected than TFIID-dependent genes, reflecting differences in their chromatin dynamics. We propose that careful tuning of Set1 levels by regulated degradation is critical for the establishment and maintenance of proper H3K4 methylation patterns. Eukaryotic genomic DNA wraps around histone octamers, assembling the nucleosomes to form the chromatin. In addition to compacting the genome, this packaging provides a mechanism for regulating DNA accessibility to proteins involved in various nuclear processes (Li and Reinberg, 2011Li G. Reinberg D. Chromatin higher-order structures and gene regulation.Curr. Opin. Genet. Dev. 2011; 21: 175-186Crossref PubMed Scopus (311) Google Scholar). A series of posttranslational modifications on histone tails are recognized by various factors involved in modulating chromatin structure and other aspects of nuclear biology (Campos and Reinberg, 2009Campos E.I. Reinberg D. Histones: annotating chromatin.Annu. Rev. Genet. 2009; 43: 559-599Crossref PubMed Scopus (638) Google Scholar). Histone H3 lysine 4 (H3K4) methylation is most commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) (Shilatifard, 2012Shilatifard A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.Annu. Rev. Biochem. 2012; 81: 65-95Crossref PubMed Scopus (674) Google Scholar). A peak of trimethylation (H3K4me3) at the promoter is followed downstream by H3K4me2 and then H3K4me1 (Rando, 2007Rando O.J. Global patterns of histone modifications.Curr. Opin. Genet. Dev. 2007; 17: 94-99Crossref PubMed Scopus (90) Google Scholar). These different levels of methylation recruit specific chromatin modifiers that include ATP-dependent remodelers, histone acetyltransferases, and histone deacetylases (Kim et al., 2012Kim T. Xu Z. Clauder-Münster S. Steinmetz L.M. Buratowski S. Set3 HDAC mediates effects of overlapping noncoding transcription on gene induction kinetics.Cell. 2012; 150: 1158-1169Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Shilatifard, 2012Shilatifard A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.Annu. Rev. Biochem. 2012; 81: 65-95Crossref PubMed Scopus (674) Google Scholar). In budding yeast, H3K4 methylation is performed by a single histone methyltransferase (HMT), Set1. Set1 and its homologs in other eukaryotes are associated with additional proteins that regulate HMT activity. The budding yeast COMPASS complex consists of Set1, Bre2 (Cps60), Sdc1 (Cps25), Spp1 (Cps40), Swd1 (Cps50), Swd2 (Cps35), Swd3 (Cps30), and Shg1 (Cps15) (Shilatifard, 2012Shilatifard A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.Annu. Rev. Biochem. 2012; 81: 65-95Crossref PubMed Scopus (674) Google Scholar). Cells lacking Swd1 or Swd3 lose all H3K4 methylation, while Sdc1 and Bre2 are important for the transition between dimethylation and trimethylation. SPP1 deletions have decreased levels of H3K4 trimethylation (Nedea et al., 2008Nedea E. Nalbant D. Xia D. Theoharis N.T. Suter B. Richardson C.J. Tatchell K. Kislinger T. Greenblatt J.F. Nagy P.L. The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes.Mol. Cell. 2008; 29: 577-587Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Swd2 is essential for viability due to its functions in RNAPII termination, but is also important for H3K4 methylation (Cheng et al., 2004Cheng H. He X. Moore C. The essential WD repeat protein Swd2 has dual functions in RNA polymerase II transcription termination and lysine 4 methylation of histone H3.Mol. Cell. Biol. 2004; 24: 2932-2943Crossref PubMed Scopus (74) Google Scholar). Various regions of Set1 have also been reported to regulate its HMT activity, including a centrally located autoinhibitory domain (Schlichter and Cairns, 2005Schlichter A. Cairns B.R. Histone trimethylation by Set1 is coordinated by the RRM, autoinhibitory, and catalytic domains.EMBO J. 2005; 24: 1222-1231Crossref PubMed Scopus (75) Google Scholar, Kim et al., 2013Kim J. Kim J.-A. McGinty R.K. Nguyen U.T.T. Muir T.W. Allis C.D. Roeder R.G. The n-SET domain of Set1 regulates H2B ubiquitylation-dependent H3K4 methylation.Mol. Cell. 2013; 49: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Through mechanisms that are still unclear, other factors, such as the PAF complex and monoubiquitination of H2BK123 (H2Bub) by the Rad6/Bre1 complex, are also critical for H3K4 methylation (Nakanishi et al., 2009Nakanishi S. Lee J.S. Gardner K.E. Gardner J.M. Takahashi Y.H. Chandrasekharan M.B. Sun Z.-W. Osley M.A. Strahl B.D. Jaspersen S.L. Shilatifard A. Histone H2BK123 monoubiquitination is the critical determinant for H3K4 and H3K79 trimethylation by COMPASS and Dot1.J. Cell Biol. 2009; 186: 371-377Crossref PubMed Scopus (94) Google Scholar). Although progress has been made in understanding COMPASS at the biochemical and structural levels (Kim et al., 2013Kim J. Kim J.-A. McGinty R.K. Nguyen U.T.T. Muir T.W. Allis C.D. Roeder R.G. The n-SET domain of Set1 regulates H2B ubiquitylation-dependent H3K4 methylation.Mol. Cell. 2013; 49: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, Takahashi et al., 2011Takahashi Y.H. Westfield G.H. Oleskie A.N. Trievel R.C. Shilatifard A. Skiniotis G. Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human.Proc. Natl. Acad. Sci. USA. 2011; 108: 20526-20531Crossref PubMed Scopus (91) Google Scholar, Trésaugues et al., 2006Trésaugues L. Dehé P.-M. Guérois R. Rodriguez-Gil A. Varlet I. Salah P. Pamblanco M. Luciano P. Quevillon-Cheruel S. Sollier J. et al.Structural characterization of Set1 RNA recognition motifs and their role in histone H3 lysine 4 methylation.J. Mol. Biol. 2006; 359: 1170-1181Crossref PubMed Scopus (44) Google Scholar), a definitive model is still lacking for how different levels of H3K4 methylation along genes are established in vivo. Two mechanisms could explain the 5′ bias of higher-level H3K4 methylation: regulators tethering Set1 to specific locations for longer periods, or Set1’s accessory factors allosterically activating Set1 at specific points along a gene (Soares and Buratowski, 2013Soares L.M. Buratowski S. Histone Crosstalk: H2Bub and H3K4 Methylation.Mol. Cell. 2013; 49: 1019-1020Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). A major hurdle in deciphering these mechanisms is that many of the COMPASS subunits reported to modulate HMT activity are also important for complex stability in vivo. Deletion of either SWD1 or SWD3 leads to loss of Set1, while removal of Spp1 or conditional deletion of SWD2 reduces its levels (Nedea et al., 2008Nedea E. Nalbant D. Xia D. Theoharis N.T. Suter B. Richardson C.J. Tatchell K. Kislinger T. Greenblatt J.F. Nagy P.L. The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes.Mol. Cell. 2008; 29: 577-587Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In this work, we analyzed a series of Set1 mutants and found that Set1 stability is regulated by multiple signals that match protein levels to proper H3K4 methylation. N-terminal regions of Set1 contain protein degradation signals, some of which overlap the previously identified autoinhibitory domain (Schlichter and Cairns, 2005Schlichter A. Cairns B.R. Histone trimethylation by Set1 is coordinated by the RRM, autoinhibitory, and catalytic domains.EMBO J. 2005; 24: 1222-1231Crossref PubMed Scopus (75) Google Scholar, Kim et al., 2013Kim J. Kim J.-A. McGinty R.K. Nguyen U.T.T. Muir T.W. Allis C.D. Roeder R.G. The n-SET domain of Set1 regulates H2B ubiquitylation-dependent H3K4 methylation.Mol. Cell. 2013; 49: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Their deletion results in higher levels of truncated but catalytically active protein. Set1 degradation is also triggered by HMT inactivating mutations or mutation of H3K4. Remarkably, unstable Set1 mutants can be rescued in trans by expression of a functional Set1 protein. Therefore, degradation of inactive mutants is not due to intrinsic instability of the proteins, but reflects a feedback mechanism whereby H3K4 methylation stabilizes Set1. These results suggest that some earlier reported effects of COMPASS subunit mutants on Set1 stability could be indirectly due to modulation of HMT activity. Overexpression of Set1 variants lacking the degradation sequences leads to aberrant H3K4 methylation patterns. Surprisingly, H3K4 methylation on SAGA- or TFIID-dependent genes responds differently to these mutations, an effect that correlates with differences in chromatin dynamics. In sum, our data show that posttranslational mechanisms that regulate Set1 protein levels are important for establishing correct H3K4 methylation patterns. Yeast COMPASS has functions that are not dependent on Set1 HMT activity (Acquaviva et al., 2013Acquaviva L. Székvölgyi L. Dichtl B. Dichtl B.S. de La Roche Saint André C. Nicolas A. Géli V. The COMPASS subunit Spp1 links histone methylation to initiation of meiotic recombination.Science. 2013; 339: 215-218Crossref PubMed Scopus (126) Google Scholar, Cheng et al., 2004Cheng H. He X. Moore C. The essential WD repeat protein Swd2 has dual functions in RNA polymerase II transcription termination and lysine 4 methylation of histone H3.Mol. Cell. Biol. 2004; 24: 2932-2943Crossref PubMed Scopus (74) Google Scholar, Zhang et al., 2005Zhang K. Lin W. Latham J.A. Riefler G.M. Schumacher J.M. Chan C. Tatchell K. Hawke D.H. Kobayashi R. Dent S.Y.R. The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation.Cell. 2005; 122: 723-734Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). However, a catalytically inactive point mutant in Set1 (e.g., H1017K; Schlichter and Cairns, 2005Schlichter A. Cairns B.R. Histone trimethylation by Set1 is coordinated by the RRM, autoinhibitory, and catalytic domains.EMBO J. 2005; 24: 1222-1231Crossref PubMed Scopus (75) Google Scholar) phenocopies a complete SET1 deletion (Terzi et al., 2011Terzi N. Churchman L.S. Vasiljeva L. Weissman J. Buratowski S. H3K4 trimethylation by Set1 promotes efficient termination by the Nrd1-Nab3-Sen1 pathway.Mol. Cell. Biol. 2011; 31: 3569-3583Crossref PubMed Scopus (46) Google Scholar). To explore this discrepancy, we analyzed recruitment of epitope-tagged Set1 on the PMA1 and RPS13 genes by chromatin immunoprecipitation (ChIP). As previously reported (Ng et al., 2003bNg H.H. Robert F. Young R.A. Struhl K. Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity.Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, Soares and Buratowski, 2012Soares L.M. Buratowski S. Yeast Swd2 is essential because of antagonism between Set1 histone methyltransferase complex and APT (associated with Pta1) termination factor.J. Biol. Chem. 2012; 287: 15219-15231Crossref PubMed Scopus (30) Google Scholar), wild-type Set1 crosslinked most strongly near the 5′ ends of both actively transcribed genes. In contrast, almost no signal was observed for the Set1 catalytic mutant H1017K (Figure 1A). Immunoblotting was used to distinguish whether loss of enzymatic activity affected Set1 protein levels or only its interaction with chromatin. Because wild-type Set1 levels are already low in extracts, proteins were first immunoprecipitated using the epitope tag and then blotted with anti-Flag antibody, a quantitative approach previously validated by Mersman et al., 2012Mersman D.P. Du H.-N. Fingerman I.M. South P.F. Briggs S.D. Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression.J. Biol. Chem. 2012; 287: 2652-2665Crossref PubMed Scopus (31) Google Scholar. These experiments showed that Set1 H1017K mutant protein was essentially undetectable (Figure 1B). This reduction was not related to expression of Set1 from a heterologous promoter or plasmid, as the same result was obtained when the SET1 H1017K allele was integrated at the chromosomal locus (Figure S1A). The same loss of Set1 was also seen with the inactivating substitution Y967A. These differences were posttranscriptional, as no changes in mRNA levels were observed between the different alleles (Figure 1B). Drastically reduced levels of Set1 have been reported with other SET domain mutants of Set1, as well as in other COMPASS mutants lacking H3K4 methylation, such as deletions of Swd1 or Swd3 subunits (Mersman et al., 2012Mersman D.P. Du H.-N. Fingerman I.M. South P.F. Briggs S.D. Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression.J. Biol. Chem. 2012; 287: 2652-2665Crossref PubMed Scopus (31) Google Scholar, Nedea et al., 2008Nedea E. Nalbant D. Xia D. Theoharis N.T. Suter B. Richardson C.J. Tatchell K. Kislinger T. Greenblatt J.F. Nagy P.L. The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes.Mol. Cell. 2008; 29: 577-587Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, Nislow et al., 1997Nislow C. Ray E. Pillus L. SET1, a yeast member of the trithorax family, functions in transcriptional silencing and diverse cellular processes.Mol. Biol. Cell. 1997; 8: 2421-2436Crossref PubMed Scopus (196) Google Scholar, Sollier et al., 2004Sollier J. Lin W. Soustelle C. Suhre K. Nicolas A. Géli V. de La Roche Saint-André C. Set1 is required for meiotic S-phase onset, double-strand break formation and middle gene expression.EMBO J. 2004; 23: 1957-1967Crossref PubMed Scopus (103) Google Scholar). Interestingly, a point mutant that only partially decreases H3K4 methylation (K1007/8A) had low but detectable amounts of Set1 protein, whereas a Y1052F substitution that increases H3K4 trimethylation (Takahashi et al., 2009Takahashi Y.H. Lee J.S. Swanson S.K. Saraf A. Florens L. Washburn M.P. Trievel R.C. Shilatifard A. Regulation of H3K4 trimethylation via Cps40 (Spp1) of COMPASS is monoubiquitination independent: implication for a Phe/Tyr switch by the catalytic domain of Set1.Mol. Cell. Biol. 2009; 29: 3478-3486Crossref PubMed Scopus (44) Google Scholar) actually had increased Set1 protein levels (Figure 1B). Therefore, in COMPASS mutants, Set1 protein levels track the levels of H3K4 methylation. The Set1, Swd1, and Swd3 mutants could affect COMPASS stability, leading to loss of H3K4 methylation. However, given the minor structural perturbations predicted for the Set1 point mutants, we considered the converse possibility that loss of methylation leads to reduced Set1 levels. Supporting this hypothesis, expression of an untagged wild-type Set1 protein, which restores normal levels of H3K4 methylation, also strongly stabilized epitope-tagged Set1 H1017K (Figure 1C). Therefore, reduced Set1 H1017K levels are not due to intrinsic instability caused by the amino acid change, or its lack of methyltransferase activity. To extend these results, other unstable mutants were coexpressed with wild-type Set1. An N-SET domain deletion that is normally degraded (Mersman et al., 2012Mersman D.P. Du H.-N. Fingerman I.M. South P.F. Briggs S.D. Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression.J. Biol. Chem. 2012; 287: 2652-2665Crossref PubMed Scopus (31) Google Scholar) was rescued by expression of wild-type Set1, as was a post-SET domain deletion mutant (Figure 1C). In contrast, a SET domain deletion was not rescued, perhaps because this region of the protein is essential for Set1’s association with the nucleosome for sensing the H3K4 methylation status (Figure 1C). To test whether H3K4 methylation affects the stability of wild-type Set1, protein levels were assayed in a strain lacking Rtf1, a PAF complex subunit required for H2Bub and H3K4 methylation (Ng et al., 2003aNg H.H. Dole S. Struhl K. The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B.J. Biol. Chem. 2003; 278: 33625-33628Crossref PubMed Scopus (219) Google Scholar). Supporting our model, this strain showed drastically reduced levels of Set1 protein (Figure 1D). At 1,080 residues, Set1 is a large protein, yet roughly the final 300 C-terminal residues alone support H3K4 methylation in vivo (Briggs et al., 2001Briggs S.D. Bryk M. Strahl B.D. Cheung W.L. Davie J.K. Dent S.Y. Winston F. Allis C.D. Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae.Genes Dev. 2001; 15: 3286-3295Crossref PubMed Scopus (474) Google Scholar). This region contains the catalytic SET domain flanked by N-SET and post-SET domains, and it mediates the interactions with all the other COMPASS subunits except Swd2 and Shg1 (Kim et al., 2013Kim J. Kim J.-A. McGinty R.K. Nguyen U.T.T. Muir T.W. Allis C.D. Roeder R.G. The n-SET domain of Set1 regulates H2B ubiquitylation-dependent H3K4 methylation.Mol. Cell. 2013; 49: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Given that the N-terminal regions are thought to regulate COMPASS activity, we asked whether they might do so via effects on Set1 stability. In comparison with the full-length protein, Set1 lacking the N-terminal 779 residues (Set1Δ780) is expressed at much higher levels, indicating that regions upstream of the N-SET domain contain degradation sequences. Despite its higher levels, Set1Δ780 has greatly reduced levels of H3K4me2 and H3K4me3 (Figure 2A). ChIP experiments suggested that this reduction may be due to decreased Set1 recruitment at promoter-proximal regions of genes (Figure 2B). Interestingly, Set1Δ780 protein was still partially degraded upon H1017K substitution or deletion of RTF1 (Figure 2C), suggesting that both N-terminal and C-terminal regions contain degradation sequences that mediate distinct mechanisms for regulating Set1 protein levels. To sense H3K4 methylation status, COMPASS must associate with chromatin. COMPASS recruitment is mediated by association with the RNAPII elongation complex, and depends on both the PAF complex and phosphorylation of the Rpb1 C-terminal domain (Krogan et al., 2003Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. et al.The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation.Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, Ng et al., 2003bNg H.H. Robert F. Young R.A. Struhl K. Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity.Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). Accordingly, we tested whether inhibition of RNAPII affects Set1 protein levels. Treating cells with 6-azauracil (6AU) or the general RNA polymerase inhibitor thiolutin strongly decreased full-length Set1 protein levels, whether expressed from a plasmid or the genomic locus (Figures 2D and S1B). In contrast, Set1Δ780 levels were slightly increased by 6AU (Figure 2D), indicating that the N-terminal regions contain sequences that destabilize Set1 in the absence of transcription. Given the role of the PAF complex in recruiting Set1 to chromatin, we tested deletions of subunits other than Rtf1. Cells without Paf1 or Ctr9 lacked both di-H3K4 and tri-H3K4 methylation, cdc73Δ showed limited H3K4me2, and leo1Δ had normal methylation (Figure 2E). Correspondingly, Set1 levels were strongly reduced in paf1Δ or ctr9Δ cells, and cdc73Δ cells had low but detectable amounts of Set1. Consistent with the model that Set1 N-terminal regions modulate protein stability based on cotranscriptional recruitment, Set1Δ780 levels were unaffected in these PAF mutants (Figure 2E). Given the sensitivity of Set1 protein stability to H3K4 methylation, we tested the effect of mutating H3K4. Set1 crosslinking to PMA1 and RPS13 was lost in an H3K4A mutant, but not when a similar change was made at H3K36 (Figure 3A). Correspondingly, H3K4 mutations strongly decreased Set1 levels, whereas H3K79 or H3K36 mutations did not (Figure 3B). Importantly, the H3K4A mutant abolished the Set1Δ780 ChIP signal (Figure 3C) without reducing its protein levels (Figure 3D), demonstrating that H3K4 is needed for Set1 recruitment. These results further indicate the existence of at least two degradation signals within Set1. Regions within the N-terminal 780 amino acids promote Set1 degradation upon transcription inhibition and in cells lacking PAF subunits that mediate COMPASS recruitment. This mechanism ensures that Set1 levels do not exceed the level needed for cotranscriptional histone methylation. The Set1 C-terminal regions mediate degradation in response to loss of H3K4 methylation, as evidenced by the instability of Set1Δ780 carrying the H1017K catalytic site mutation or in combination with rtf1Δ. Interestingly, combining H3K4A or an H3 tail deletion (Δ4-30) with the unstable Set1Δ780(H1017K) mutant stabilized the protein, a result that was not seen with full-length Set1 (Figure 3E). This finding makes sense if one assumes that COMPASS must first be recruited before it can sense the status of histone methylation. To better map the Set1 regions that limit its protein levels, we deleted various stretches of the protein (Figure 4A; see Figure S2A for a schematic of the mutants). Within the first 700 amino acids, individual deletions of 100 residues did not significantly increase Set1 protein levels (Figure 4A). However, levels of the Δ100-200 and Δ200-300 deletions were strongly decreased, correlating with loss of H3K4 methylation, particularly H3K4me2 (Figure 4A). Residues 100–200 are necessary for interaction with Swd2 (Kim et al., 2013Kim J. Kim J.-A. McGinty R.K. Nguyen U.T.T. Muir T.W. Allis C.D. Roeder R.G. The n-SET domain of Set1 regulates H2B ubiquitylation-dependent H3K4 methylation.Mol. Cell. 2013; 49: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and the 200–300 deletion removes part of the first RNA recognition motif (RRM) (Schlichter and Cairns, 2005Schlichter A. Cairns B.R. Histone trimethylation by Set1 is coordinated by the RRM, autoinhibitory, and catalytic domains.EMBO J. 2005; 24: 1222-1231Crossref PubMed Scopus (75) Google Scholar), and both were previously shown to be important for H3K4 methylation. Interestingly, Δ100, Δ300-400, and Δ600-700 also produced less H3K4me3 without reducing Set1 levels, suggesting that H3K4me2 may be sufficient for Set1 stabilization (Figure 4A). The observation that none of the small deletions increased Set1 to the levels seen with Set1Δ780 suggested the presence of multiple redundant degradation signals in the N-terminal region of Set1. In support of this idea, independent expression of individual 100 residue stretches showed that all were degraded, except for a region between residues 500 and 600 (Figure S2C). Given the lack of protein stabilization by small deletions, we proceeded to truncate Set1 from the N terminus in 100 residue increments (Figures 4B and S2A). As seen with the internal deletions, truncations that removed residues between amino acid 100 and 300 decreased Set1 levels and H3K4 methylation. Importantly, even the most unstable mutants (Δ300 and Δ400) could be rescued by coexpression of wild-type Set1 (Figure S2C), demonstrating that these truncations were not intrinsically unstable but were degraded in response to loss of H3K4 methylation. Truncations past amino acid 400 gradually led to increased protein levels and rescue of H3K4 methylation (Figure 4B). The levels of Δ500 were roughly equal to those of the wild-type protein, and further truncations resulted in much higher levels of Set1. Maximal Set1 amounts were observed with Set1Δ700, which also restored bulk H3K4 methylation. A comparison of the levels of Set1Δ700 expressed from an overexpression plasmid or the endogenous chromosomal locus shows that the increase was intrinsic to the deletion (Figure S2D). As with Set1Δ780, the protein levels of the highly expressed truncations were still reduced by the catalytic mutation H1017K (Figure S2E). The Set1 N-terminal truncation series was tested for its ability to complement a SET1 deletion (Figure 4C). The ability to restore growth on 6AU tracked closely with protein expression and restoration of H3K4 methylation. One important function of H3K4 methylation in yeast is to modulate the kinetics of gene induction, particularly in response to carbon source shifts (Kim et al., 2012Kim T. Xu Z. Clauder-Münster S. Steinmetz L.M. Buratowski S. Set3 HDAC mediates effects of overlapping noncoding transcription on gene induction kinetics.Cell. 2012; 150: 1158-1169Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, Margaritis et al., 2012Margaritis T. Oreal V. Brabers N. Maestroni L. Vitaliano-Prunier A. Benschop J.J. van Hooff S. van Leenen D. Dargemont C. Géli V. Holstege F.C. Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3′-end antisense transcription.PLoS Genet. 2012; 8: e1002952Crossref PubMed Scopus (89) Google Scholar). Although two catalytic point mutants showed altered response kinetics similar to those previously seen upon SET1 deletion, Set1Δ700 responded like wild-type Set1 at most, but not all, genes tested (Figure 4D). Therefore, the overexpressed truncation protein can largely substitute for the wild-type, but shows gene-specific effects. Although the deeper Set1 truncations restored wild-type methylation levels as assayed by immunoblotting of whole-cell extracts, we tested all of our mutants for proper methylation patterns along the PMA1 and RPS13 genes by ChIP. Consistent with immunoblotting, H3K4 methylation was virtually absent in Set1Δ200-300 and was normal in Set1Δ400-500 and Set1Δ500-600 (Figure 4E). Remarkably, the remaining deletions affected H3K4 methylation differently on the two genes. Both H3K4me3 and H3K4me2 in Set1Δ100, Δ100-200, Δ300-400, and Δ600-700 were nearly abolished on PMA1 (Figure 4E). In contrast, the methylation levels on RPS13 were the same or even increased in most mutants, with a pronounced 5′ shift of the H3K4me2 peak. Therefore, N-terminal regions of Set1 are needed for proper H3K4 methylation patterns. The differential effects on PMA1 and RPS13 were not restricted to Set1 mutants, as similar changes occurred with other mutations that reduced, but did not abolish, H3K4 methylation. These included H3R2 mutations, deletion of SAGA subunits, or deletion of other COMPASS components (Figures S3A and S3B). Therefore, the gene-specific changes in methylation reflect a general response to reduced HMT activity rather than functions of particular Set1 domains. The Set1 N-terminal truncation series showed similar differential effects, but with important differences (Figure 4E). All truncations had reduced H3K4me3 on PMA1, but only Set1Δ300, the one mutant that showed markedly lower protein levels, affected RPS13. The H3K4me2 peak was shifted toward the RPS13 promoter in all mutants that truncated beyond the first 100 amino acids. In the highly expressed Set1Δ600 and Δ700 mutants, the H3K4me2 signal was restored at PMA1 and spread further downstream throughout the transcribed regions of RPS13 (Figure 4E). These results argue that the N-terminal truncations reduce Set1 activity, but the increased protein lev" @default.
• W2051163721 created "2016-06-24" @default.
• W2051163721 creator A5048199254 @default.
• W2051163721 creator A5053312726 @default.
• W2051163721 creator A5056530950 @default.
• W2051163721 creator A5073459416 @default.
• W2051163721 creator A5079482663 @default.
• W2051163721 date "2014-03-01" @default.
• W2051163721 modified "2023-10-16" @default.
• W2051163721 title "Feedback Control of Set1 Protein Levels Is Important for Proper H3K4 Methylation Patterns" @default.
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