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. Author manuscript; available in PMC: 2014 Apr 25.
Published in final edited form as: Cell Rep. 2014 Mar 6;6(6):961–972. doi: 10.1016/j.celrep.2014.02.017

Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns

Luis M Soares 1, Marta Radman-Livaja 2,3, Sherry G Lin 1, Oliver J Rando 2, Stephen Buratowski 1,*
PMCID: PMC3999964  NIHMSID: NIHMS569506  PMID: 24613354

Abstract

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 auto-inhibitory domain, as well as the catalytic domain. Truncation mutants of Set1 that cause under- or over-expression 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 establishment and maintenance of proper H3K4 methylation patterns.

Introduction

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, 2011). A series of post-translational modifications on histone tails are recognized by various factors involved in modulating chromatin structure and other aspects of nuclear biology (Campos and Reinberg, 2009). Histone H3 lysine 4 (H3K4) methylation is most commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) (Shilatifard, 2012). A peak of tri-methylation (H3K4me3) at the promoter is followed downstream by H3K4me2 and then H3K4me1 (Rando, 2007). These different levels of methylation recruit specific chromatin modifiers that include ATP-dependent remodelers, histone acetyltransferases, and histone deacetylases (Kim et al., 2012; Shilatifard, 2012).

In budding yeast, H3K4 methylation is performed by a single histone methyltransferase (HMT), Set1. Set1 and its homologues 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, 2012). Cells lacking Swd1 or Swd3 lose all H3K4 methylation, while Sdc1 and Bre2 are important for transition between di- and tri- methylation. SPP1 deletions have decreased levels of H3K4 tri-methylation (Nedea et al., 2008). Swd2 is essential for viability due to its functions in RNAPII termination, but is also important for H3K4 methylation (Cheng et al., 2004). Various regions of Set1 have also been reported to regulate its HMT activity, including a centrally located auto-inhibitory domain (Schlichter and Cairns, 2005; Kim et al., 2013). 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., 2009).

While progress has been made in understanding COMPASS at the biochemical and structural levels (Kim et al., 2013; Takahashi et al., 2011; Trésaugues et al., 2006), 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, 2013). 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., 2008).

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 auto-inhibitory domain (Schlichter and Cairns, 2005; Kim et al., 2013). 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 expressing 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 post-translational mechanisms regulating Set1 protein levels are important for establishing correct H3K4 methylation patterns.

Results

Set1 is stabilized by H3K4 methylation and transcription

Yeast COMPASS has functions that are not dependent on Set1 HMT activity (Acquaviva et al., 2013; Cheng et al., 2004; Zhang et al., 2005). However, a catalytically inactive point mutant in Set1 (e.g., H1017K; Schlichter and Cairns, 2005) phenocopies a complete SET1 deletion (Terzi et al., 2011). To explore this discrepancy, recruitment of epitope-tagged Set1 on the PMA1 and RPS13 genes was analyzed by chromatin immunoprecipitation (ChIP). As previously reported, wild type Set1 crosslinked most strongly near 5' ends of both actively transcribed genes. In contrast, almost no signal was observed for the Set1 catalytic mutant H1017K (FIG 1A).

Figure 1. Set1 protein levels correlate with H3K4 methylation.

Figure 1

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A) ChIP of wild-type or H1017K Set1 to PMA1 and RPS13. Flag-tagged wild type or H1017K Set1 was immunoprecipitated from chromatin and associated DNA analyzed by PCR. Upper panel shows schematic of genes, with bars indicating PCR products. Fold enrichment was calculated by normalization against values for untagged strain. Error bars represent standard error from biological triplicates. B) Flag-Set1 protein and mRNA levels. Extracts from set1Δ strains transformed with plasmids expressing Flag-tagged wild type Set1 (lane 1), H1017K (lane 2), Y967A (lane 3), K1007/8A (lane 4) or Y1052F (lane 5). Set1 substitutions were immunoprecipitated with M2-agarose beads, resolved by SDS-PAGE and analyzed by immunoblotting for the Flag-tag. Asterisk marks a cross-reacting band that serves as an internal loading control. Separately, 1/10 of the extract used for immunoprecipitation was resolved by SDS-PAGE and analyzed by blotting for TBP (as an input control), H3K4me3, H3K4me2 and Histone H3. At bottom, 1 µg of RNA from the strains was analyzed by RT-PCR with primers specific for SET1 or ADH1, -RT is a control reaction lacking reverse transcriptase. C) Stabilization of Set1 mutants by wildtype Set1. Flag-tagged wild-type (lanes 2, 4 and 6), H1017K (lanes 3, 5 and 7), ΔNSET (lanes 8 and 11), ΔSET (lanes 9 and 12) or ΔPSET (lanes 10 and 13) Set1 was expressed and immunoprecipitated from set1Δ (lanes 1–3 and 6–10) or SET1 (lanes 4, 5 and 11–13) strains and analyzed as in part B. D) Flag-Set1 protein levels in RTF1 deletion. Extracts were prepared from set1Δ (lanes 1 and 2) or set1Δ, rtf1Δ (lanes 3 and 4) cells expressing wild type (lanes 1 and 3) or H1017K (lanes 2 and 4) Flag-tagged Set1 and analyzed as in panel B.

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. (2012). These experiments showed that Set1 H1017K mutant protein was essentially undetectable (FIG 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 (FIG S1A). The same loss of Set1 was also seen with the inactivating substitution Y967A. These differences are post-transcriptional, as no changes in mRNA levels were observed between the different alleles (FIG 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., 2012; Nedea et al., 2008; Nislow et al., 1997; Sollier et al., 2004). Interestingly, a point mutant that only partially decreases H3K4 methylation (K1007/8A) has low but detectable amounts of Set1 protein, while a Y1052F substitution that increases H3K4 trimethylation (Takahashi et al., 2009) actually has increased Set1 protein levels (FIG 1B). Therefore, in COMPASS mutants, Set1 protein levels track 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 wildtype Set1 protein, which restores normal levels of H3K4 methylation, also strongly stabilized epitope-tagged Set1 H1017K (FIG 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 co-expressed with wild-type Set1. An N-SET domain deletion that is normally degraded (Mersman et al., 2012) is rescued by expression of wild-type Set1, as is a post-SET domain deletion mutant (FIG 1C). In contrast, a SET domain deletion was not rescued, perhaps because this region of the protein is essential for Set1 association with the nucleosome for sensing the H3K4 methylation status (FIG 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., 2003a). Supporting our model, this strain shows drastically reduced levels of Set1 protein (FIG 1D).

At 1080 residues, Set1 is a large protein, yet roughly the final 300 C-terminal residues alone support H3K4 methylation in vivo (Briggs et al., 2001). 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., 2013). We asked whether the N-terminal regions, thought to regulate COMPASS activity, might do so via effects on Set1 stability. In comparison to the fulllength 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 me3 (FIG 2A). ChIP experiments suggest this reduction may be due to decreased Set1 recruitment at promoter-proximal regions of genes (FIG 2B). Interestingly, Set1Δ780 protein is still partially degraded upon H1017K substitution or deletion of RTF1 (FIG 2C), suggesting that both N-terminal and C-terminal regions contain degradation sequences mediating distinct mechanisms for regulating Set1 protein levels.

Figure 2. Independent degradation signals in the Set1 N-terminal and C-terminal domains.

Figure 2

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A) Deletion of the N-terminal 780 amino acids increases Set1 protein levels. Flag-tagged Set1 was analyzed by immunoprecipitation as in FIG 1; wild type (lanes 1), H1017K (lane 2), or Set1Δ780 (lane 3). Lanes 4 (wild type) and 5 (Set1Δ780) show input extract immunoblotted for H3K4 methylation. B) ChIP of Set1Δ780. Flag-tagged wild-type or Δ780 Set1 recruitment to PMA1 and RPS13 was analyzed as in FIG 1A. Error bars represent standard error from biological triplicates. C) H1017K substitution and rtf1Δ reduce Flag-Set1Δ780 protein levels. Extracts from the indicated cells were analyzed as in previous figures: Flag-tagged Set1 Δ780 (lanes 1, 3, and 4), Δ780 H1017K (lane 2), rtf1Δ (lane 4). D) Set1 protein levels are reduced upon transcription inhibition only in the context of full-length protein. Extracts from cells with the indicated genotypes were analyzed as above. Left panel shows results from cells expressing full length Set1 while right panel shows cells expressing Set1Δ780. Where indicated, cells were grown for 30 minutes in the presence of 500 µg/ml of 6AU (lanes 4, 7, and 8), 3 µg/ml of thiolutin (lane 3), or DMSO as a negative control (lanes 1, 2, 5, and 6). E) Set1Δ780 still requires PAF complex for H3K4 methylation but not protein stability. Immunoblot analysis was performed as in previous figures for cells expressing Flag-tagged full-length Set1 (lanes 1–5) or Set1Δ780 (lanes 6–10) in paf1Δ (lanes 2 and 7), ctr9Δ (lanes 3 and 8), cdc73Δ (lanes 4 and 9), or leo1Δ (lanes 5 and 10) background. 1/10 of input extracts were analyzed for TBP, H3K4me3, H3K4me2, and histone H3.

To sense H3K4 methylation status, COMPASS must associate with chromatin. COMPASS recruitment is mediated by association with the RNAPII elongation complex, dependent upon both the PAF complex and phosphorylation of the Rpb1 C-terminal domain (Krogan et al., 2003; Ng et al., 2003b). 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 (FIG 2D and S1B). In contrast, Set1Δ780 levels are slightly increased by 6AU (FIG 2D), indicating that the N-terminal regions contain sequences that destabilize Set1 in the absence of transcription.

Given the role of PAF complex in recruiting Set1 to chromatin, deletions of the subunits other than Rtf1 were tested. Cells without Paf1 or Ctr9 lack both di- and tri- H3K4 methylation, cdc73Δ shows limited H3K4me2, and leo1Δ has normal methylation (FIG 2E). Correspondingly, Set1 levels are strongly reduced in paf1Δ or ctr9Δ cells, while cdc73Δ cells have low but detectable amounts of Set1. Consistent with the model that Set1 N-terminal regions modulate protein stability based on co-transcriptional recruitment, Set1Δ780 levels are unaffected in these PAF mutants (FIG 2E).

Given the sensitivity of Set1 protein stability to H3K4 methylation, the effect of mutating H3K4 was tested. Set1 crosslinking to PMA1 and RPS13 was lost in an H3 K4A mutant, but not when a similar change is made at H3K36 (FIG 3A). Correspondingly, H3K4 mutations strongly decreased Set1 levels, whereas H3K79 or H3K36 mutations did not (FIG 3B). Importantly, the H3K4A mutant abolishes the Set1Δ780 ChIP signal (FIG 3C) without reducing its protein levels (Fig 3D), demonstrating that H3K4 is needed for Set1 recruitment.

Figure 3. H3K4 contributes to Set1 recruitment.

Figure 3

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A) ChIP of Flag-tagged Set1 to PMA1 and RPS13 in wild type H3K4, H3K4A, or H3K36A strains. Procedure and PCR primer pairs were as described in FIG 1A. Error bars represent standard error from biological triplicates. B) Set1 protein and RNA levels in histone H3 substitution strains were analyzed as in FIG 1B. Flag-tagged Set1 was immunoprecipitated from wild-type H3 (lane 1), K4R (lane 3), K4A (lane 4), K4Q (lane 5), K36R (lane 6), K36A (lane 7), or K79A (lane 8) cell extracts. Lane 2 shows cells lacking Set1. Lower band marked by asterisk is a cross-reacting protein that serves as an internal loading control. C) Effects of H3K4 substitution in the recruitment of Set1 N-terminal Δ780 truncation to active genes. ChIP of Flag-tagged Set1 wild type or Δ780 N-terminal truncation to PMA1 and RPS13 in wild type H3K4 or H3K4A strains. Indicated Flag-tagged Set1 proteins, expressed in set1Δ strains containing histone H3K4 or an H3K4A substitution, were immunoprecipitated and associated DNA was analyzed by PCR. D) Effects of H3K4A substitution on Set1Δ780 truncation protein levels. Extracts from set1Δ strains in background containing wild type (lanes 1 and 3) or K4A substitution (lanes 2 and 4) in histone H3 transformed with plasmids expressing Flag-tagged wild type Set1 (lanes 1 and 2) or Flag-tagged Set1 lacking the N-terminal first 780 residues (Flag-Set1Δ780, lanes 3 and 4) were analyzed as in previous figures. O.E. refers to indicated region of the same membrane exposed for a longer time. 1/10 of the extract used for immunoprecipitation was resolved by SDS-PAGE and analyzed by western blot against TBP as a loading control. E) Differential response of full length and Δ780 Set1 to H3 mutations and H1017K substitution. Immunoprecipitated Flag-tagged Set1 from the indicated cells was analyzed as above.

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 don't exceed that needed for co-transcriptional 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 H3 K4A or an H3 tail deletion (Δ4–30) with the unstable Set1Δ780(H1017K) mutant stabilizes the protein, a finding not seen with full length Set1 (FIG 3E). This finding makes sense if one assumes that COMPASS must first be recruited before it can sense the status of histone methylation.

Mapping Set1 degradation regions

To better map the Set1 regions that limit its protein levels, we deleted various stretches of the protein (FIG 4A, see FIG S2A for schematic of mutants). Within the first 700 amino acids, individual deletions of 100 residues did not significantly increase Set1 protein levels (FIG 4A). However, levels of the Δ100–200 and Δ200–300 deletions were strongly decreased, correlating with loss of H3K4 methylation, particularly H3K4me2 (FIG 4A). Residues 100–200 are necessary for interaction with Swd2 (Kim et al., 2013) and the 200–300 deletion removes part of the first RRM (Schlichter and Cairns, 2005), 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 (FIG 4A).

Figure 4. Mapping and characterization of Set1 deletion mutations affecting stability.

Figure 4

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A) Effects of 100 amino acid internal deletions on Set1 protein levels and H3K4 methylation. Extracts from cells expressing wild type (lane 1) or indicated deletions of Flag-tagged Set1 were analyzed by immunoblotting as in previous figures. B) Effects N-terminal truncations of Set1 on protein levels and H3K4 methylation. Extracts from set1Δ strains transformed with wild type (lane 1) or the indicated N-terminal truncations were analyzed as above. C) Effects of Set1 truncations on 6AU resistance. Serial 1:3 dilutions of indicated strains were spotted on synthetic complete (SC) plates with or without 100 µg/ml 6AU. D) Effects of Set1 mutants on gene induction kinetics. Strains with the indicated genotypes were grown in raffinose (RA) media, shifted to galactose and then glucose for the indicated times. RNA from the indicated strains was analyzed by RT-PCR and quantitated by normalization to an ACT1 control. E) ChIP patterns of H3K4me3 and H3K4me2 at PMA1 and RPS13 genes in Set1 deletions/truncations. ChIP of H3K4me3 and H3K4me2 methylation in PMA1 and RPS13 genes in strains used in FIG 4 and B. Numbered bars (inset) designate PCR product locations as depicted in FIG 1A.

The observation that none of the small deletions increased Set1 to levels seen with Set1Δ780 suggested multiple redundant degradation signals in the N-terminal region of Set1. In support of this idea, independent expression of individual 100 residue stretches shows all are degraded except for a region between residues 500 and 600 (FIG S2C). Given the lack of protein stabilization by small deletions, we proceeded to truncate Set1 from the N-terminus in 100 residue increments (FIG 4B, S2A). As seen with the internal deletions, truncations removing residues between amino acid 100 and 300 decrease Set1 levels and H3K4 methylation. Importantly, even the most unstable mutants (Δ300 and Δ400) can be rescued by co-expression of wild-type Set1 (FIG S2C), demonstrating that these truncations are not intrinsically unstable but are degraded in response to loss of H3K4 methylation.

Truncations past amino acid 400 gradually lead to increased protein levels and rescue of H3K4 methylation (FIG 4B). Levels of Δ500 are roughly equal to the wild-type protein, and further truncations result in much higher levels of Set1. Maximal Set1 amounts are observed with Set1Δ700, which also restores bulk H3K4 methylation. Comparison of Set1Δ700 levels expressed from an overexpression plasmid or the endogenous chromosomal locus shows that the increase is intrinsic to the deletion (FIG S2D). As with Set1Δ780, the protein levels of the highly expressed truncations are still reduced by the catalytic mutation H1017K (FIG S2E).

The Set1 N-terminal truncation series was tested for the ability to complement a SET1 deletion (FIG 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., 2012; Margaritis et al., 2012). While 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 (FIG 4D). Therefore, the overexpressed truncation protein can largely substitute for wild-type, but shows gene-specific effects.

Differential effects of Set1 mutations on SAGA and TFIID genes

Although the deeper Set1 truncations restore wild-type methylation levels as assayed by immunoblotting of whole cell extracts, we tested all 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 (FIG 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 (FIG 4E). In contrast, 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 reduce, but do not abolish, H3K4 methylation. These include H3R2 mutations, deletion of SAGA subunits, or deletion of other COMPASS components (FIG S3A, B). 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 (FIG 4E). All truncations have reduced H3K4me3 on PMA1, while only Set1Δ300, the one mutant showing markedly lower protein levels, affects RPS13. The H3K4me2 peak is shifted towards the RPS13 promoter in all mutants that truncate beyond the first 100 amino acids. In the highly expressed Set1Δ600 and Δ700 mutants, the H3K4me2 signal is restored at PMA1 and spreads further downstream throughout the transcribed regions of RPS13 (FIG 4E). This results argue that the N-terminal truncations reduce Set1 activity, but that increased protein levels of the deeper deletions can compensate for this effect at some genes.

Interestingly, truncating the first 400 to 600 residues of Set1 nearly abolished H3K4me3 in whole cell extracts (FIG 4B) and on PMA1, yet these strains have normal levels of H3K4me3 at RPS13 (FIG 4E). The hypomorphic K1007/8A mutant (FIG 1B) behaved similarly (FIG S3C). ChIP experiments with the hyperactive Y1052T substitution, which has higher total H3K4me3 levels (FIG 1B), showed increased H3K4me3 at PMA1 but not RPS13 (SUP FIG S3C). These results show that Set1 mutants have gene-specific effects and that general conclusions cannot be drawn from immunoblotting alone.

Given the differential responses of PMA1 and RPS13 to reduced Set1 activity, we sought to identify features distinguishing these genes. Huisinga and Pugh (2004) sorted genes into two classes based on sensitivity to TFIID or SAGA mutants. TFIID function predominates at approximately 90% of genes, mostly constitutively expressed. Meanwhile SAGA is important for expression of the remaining, often highly regulated, 10%. PMA1 is classified as SAGA-dependent, whereas RPS13 is TFIID responsive.

To test whether the SAGA-TFIID distinction correlated with differential Set1 response, H3K4 methylation was assayed genome-wide by ChIP-chip analysis. In a wild-type Set1 strain, H3K4me2 and me3 levels are higher on average over SAGA-dependent genes (FIG 5A), supporting the assertion that these genes contribute significantly to the immunoblotting signals in whole cell extracts. Confirming the single gene results, strains carrying Set1Δ500 or Δ700 N-terminal truncations cause reduction of both tri- and dimethylation peaks on SAGA-dependent genes (FIG 5B). In contrast, H3K4me3 is far less affected at TFIID-dependent genes (FIG 5B). Set1 truncations also produce a marked shift of H3K4me2 towards the transcription start site of TFIID-dependent promoters, as observed at RPS13. Interestingly, regions distant from gene promoters, and therefore not normally methylated, show increased H3K4me2 and me3, suggesting that the high levels of these truncated proteins (FIG 4B) may promiscuously methylate H3K4 at increased levels throughout the genome.

Figure 5. H3K4 methylation shows differential behavior on SAGA- versus TFIID-dependent genes.

Figure 5

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A) Metagene analysis from whole genome profiling of H3K4me3 and H3K4me2 levels in SAGA versus TFIID dependent genes. B) Metagene analysis of H3K4me3 and H3K4me2 in wild-type Set1, Set1Δ500, or Set1Δ700 strains was plotted for SAGA and TFIID dependent genes after subtraction of the set1Δ background signal. C) Upper panel shows immunoblots of extracts from strains carrying "anchor-away" alleles (FRB fusions) of Bre2, Shg1, or Swd1, both before and after 5 hour rapamycin treatment. Blots were probed for H3K4me3, H3K4me2, and total H3. Lower panel shows normalized ChIP results for H3K4me3 at the indicated promoters during a time course of Swd1-FRB depletion. See Fig S4 for additional related results.

Differential effects of Set1 depletion on SAGA and TFIID genes

We sought to determine if the different behavior of SAGA and TFIID genes was specifically due to differential requirements for the N-terminal regions, or was instead an intrinsic response of the promoters to lower Set1 activity. COMPASS complex activity was depleted using the "anchor-away" technique, where a domain of FK506-rapamycinbinding protein (FRB) is fused to the protein of interest and, upon rapamycin treatment, trapped in the cytoplasm via an interaction with an FKBP-ribosomal protein fusion (Haruki et al., 2008). Of three COMPASS fusions tested (Bre2, Shg1, and Swd1), Swd1-FRP produced the most complete loss of H3K4 methylation (FIG 5C). Through the depletion time course, H3K4 methylation was monitored by ChIP at three SAGAdependent (ECM33, PYK1, and PMA1) and three TFIID-dependent promoters (RPS13, ACT1, and YEF3). Whereas H3K4me3 dropped rapidly at SAGA-dependent genes, methylation over the TFIID-dependent promoters was largely stable (FIG 5C and S4A). This differential effect might reflect more rapid demethylation, but is most likely due to more rapid histone replacement at SAGA-dependent genes (Dion et al., 2007; Radman-Livaja et al., 2011). Supporting this idea, H4K4me3 turnover at PMA1 is transcription-dependent, as treatment of cells with 6AU before Swd1 depletion prevents loss of H3K4 methylation (SUP FIG S4B). We propose that full Set1 activity is needed to maintain H3K4 methylation at SAGA-dependent genes in the face of rapid nucleosome turnover.

Discussion

The yeast Set1 complex serves as an excellent model for the multiple Set1/MLL complexes in metazoans (Shilatifard, 2012). A key question is how these enzymes are regulated to generate the standard pattern of H3K4 methylation along active genes. Here we show that Set1 protein levels are linked to ongoing transcription and H3K4 methylation levels through feedback control of degradation. This mechanism limits Set1 levels to establish and maintain H3K4 methylation. Mutations that abrogate this regulation, or partial depletion of Set1, disrupt normal H3K4 methylation patterns. Surprisingly, SAGA-dependent genes are more strongly affected than TFIID-dependent promoters, most likely reflecting the faster nucleosome turnover rates at SAGA-dependent promoters.

Multiple regions of Set1 can trigger degradation. Sequences within the N-terminal 780 amino acids reduce Set1 levels in response to inhibition of transcription (FIG 2D), loss of the PAF complex (FIG 2E), or mutation of H3K4 (FIG 3B, D). Deletion of the first 600–780 residues leads to much higher levels of Set1 protein expression (FIG 2A, 3D, 4B). However, the overexpressed C-terminal fragment, which includes the N-SET, SET, and post-SET domains, is still down-regulated in response to mutations that disrupt Set1 catalytic activity (FIG 2C). Remarkably, with the exception of the SET domain deletion, all of the unstable Set1 mutant proteins are rescued by co-expression of wild-type Set1 (FIG 1C, S2C). Therefore, the drop in protein levels is not due to intrinsic instability of the mutants, but instead reflects a feedback mechanism that destabilizes Set1 upon reduction of transcription or H3K4 methylation (FIG S4C). Preferential degradation of Set1 not bound to RNAPII or methylated H3K4 may suppress promiscuous methylation and help maintain and reinforce the transcription-generated H3K4 methylation pattern.

Several other groups also analyzed deletion mutants of Set1. Schlichter and Cairns (2005) isolated a SET1 allele lacking the first 762 residues as a suppressor of a rsc2 mutation, but found that a smaller deletion mutant lacking just the first RRM could not suppress. Because the deeper N-terminal deletion had high levels of H3K4me3 but the smaller did not, they proposed that the RRM region counteracted an auto-inhibitory domain located upstream of the N-SET domain. However, Set1 levels were not tested and the "auto-inhibitory" domain overlaps the degradation signals we mapped. Kim et al. (2013) also analyzed a series of Set1 N-terminal deletions. In vitro experiments using truncation proteins provided evidence for auto-inhibition, as deletion of residues between residues 569 and 762 increased methyltransferase activity. However, the authors noted that discrepancies between those results and their in vivo experiments suggested further modulation, which we suspect involves control of Set1 protein levels. The fact that both auto-inhibition and degradation sequences map to the N-terminal region underscores the connection between Set1 activity and its protein levels.

Our findings are relevant to earlier studies looking at the functions of other COMPASS subunits. For example, deletion of the COMPASS subunits Swd1 or Swd3 results in loss of Set1 in vivo, which was interpreted as these subunits being important for COMPASS integrity (Halbach et al., 2009). Yet COMPASS can be assembled in vitro without Swd1 or Swd3, forming a complex with reduced methyltransferase activity (Kim et al., 2013). Therefore, in vivo degradation of Set1 in these mutants may be an indirect effect of losing H3K4 methylation. Conversely, when characterizing mutations that specifically affect specific methylation states, it becomes pertinent to ask whether the effects are directly on Set1 catalytic activity or Set1 levels, since these can produce similar results.

How is Set1 stabilized upon interaction with transcription elongation complexes or methylated chromatin? One likely possibility is that recruitment to transcribing genes physically separates Set1 from degradation factors. Alternatively, recruitment of COMPASS to chromatin could elicit placement or removal of specific post-translational modifications that modulate Set1 activity and degradation.

To ask whether Set1 degradation mechanisms were physiologically important, the pattern of H3K4 methylation along active genes was mapped in N-terminal truncation strains with either reduced or increased levels of expression (FIG 4 and 5). Not only were methylation patterns altered, but we discovered gene-specific responses that correlate with promoter dependence upon TFIID or SAGA. Deletions that strongly reduce Set1 levels essentially abolish H3K4 methylation on the SAGA-dependent PMA1 gene (FIG 4). In contrast, there was little or no reduction in methylation on TFIID-dependent RPS13, showing instead a 5' shift in the H3K4me2 peak. By immunoblotting alone, many of these mutants would be characterized as defective in higher-level methylations (FIG 4), and yet they function quite well on RPS13, so care must be taken in interpreting and extrapolating other studies where only global methylation levels are assayed. Genome-wide ChIP analysis in Set1Δ500 and Set1Δ700 strains, with normal and high protein levels, respectively, showed similar differential effects on SAGA versus TFIID genes (FIG 5A). SAGA genes showed strong loss of H3K4me2 and me3, while TFIID genes had both normal H3K4me3 and strong H3K4 dimethylation peaks shifted upstream. These mutants also have increased downstream methylation, perhaps due to spurious, non-targeted enzyme activity.

The differential methylation response of SAGA- versus TFIID-dependent genes is also seen with Set1 catalytic mutants having reduced activity, other COMPASS subunit deletions, H3R2 mutants, and SAGA mutants (FIG S3). Therefore, these results likely result from lower COMPASS activity coupled to differences in chromatin dynamics, rather than COMPASS mutants functioning differently at the two classes of genes. To test this idea, COMPASS activity supported by wild type Set1 was rapidly depleted using the "anchors-away" technique and H3K4me3 analyzed on a representative set of promoters (FIG 5C). Methylation is lost at a much faster rate on SAGA versus TFIID genes, similar to the effects seen with mutant strains. This difference may be due to faster demethylation, or more likely, the faster turnover of nucleosomes at these promoters (Dion et al., 2007; Radman-Livaja et al., 2011)

Careful control of Set1 levels may serve several purposes. First, disruption of proper feedback affects proper kinetics of gene expression changes (FIG 4D), which may be mediated by the H3K4me3/me2 gradient and overlapping non-coding transcription (Kim et al., 2012; Margaritis et al., 2012). A second possibility is to ensure that high levels of Set1 do not ectopically trigger its functions in telomeric regulation, chromosome segregation, or meiosis (Briggs et al., 2001; Kim et al., 2013; Nislow et al., 1997; Roguev et al., 2001; Sollier et al., 2004; Zhang et al., 2005). These processes presumably have their own mechanisms for affecting COMPASS stability and targeting. We note that co-transcriptional recruitment of the H3K36 HMT Set2 also prevents its degradation (Fuchs et al., 2012). Therefore, degradation of Set1 and Set2 not associated with transcription complexes may be a common strategy to regulate their levels and co-transcriptional methylation patterns.

It remains to be seen whether the mechanisms described here for yeast Set1 are conserved in higher eukaryotic enzymes. Recent reports (Tate et al., 2010; Wang et al., 2012) show that levels of Set1 homologues are also affected by post-translational mechanisms, and translocations that separate the N- and C-terminal regions of MLL1 are associated with aggressive acute leukemias (Shilatifard, 2012). It seems likely that yeast will again prove to be a valuable model for understanding all eukaryotic H3K4 methylation in both physiological and pathological conditions.

Materials and Methods

Yeast strains and plasmids

Yeast culture was performed using standard methods. Yeast strains used are listed in Supp. Table S1 and plasmids in Supp. Table S2. Treatment with 6-azauracil and thiolutin was performed with 500 µg/ml and 3 µg/ml respectively for 45 minutes. Set1 constructs were obtained by inverse PCR of PRS416-ADHp-Flag-tag-Set1 (Fingerman et al., 2005). Anchor-away assays were performed as described by Haruki et al., 2008. Genomic copies of BRE2, SHG1, and SWD1 were tagged with N-terminal FRB in strain HHY168 and anchor-away assays were performed as described by Haruki et al., 2008. Carbon source shift experiments were performed as described in Kim et al., 2012.

Antibodies

This study uses anti-H3 (Abcam Ab1791), anti-H3K4me2 (Upstate 06-030), anti-H3K4me3 (Upstate 05-745), anti-H4K4me1 (Upstate 07-436), anti-Flag (Sigma F3165), anti-TBP.

Chromatin immunoprecipitations

Chromatin immunoprecipitations were done as previously described (Soares and Buratowski, 2012). Details of the procedures are described in Supplemental Experimental Procedures.

Immunoprecipitation and Immunoblotting

Whole cell extracts were prepared as described by Fingerman et al. 2005. Experimental details are included in Supplemental Experimental Procedures.

RNA analysis

RNA was extracted from cells with hot water-equilibrated phenol. First-strand cDNA was prepared using 1 µg total RNA treated with DNAse I, Superscript II reverse transcriptase (Invitrogen), and gene specific primers indicated in Supp. Table S3. One quarter of the cDNA was used for PCR quantitation (19 cycles).

Micrococcal nuclease digestion and microarray analysis of chromatin immunoprecipitation (ChIP)

Details of the procedures are described in Supplemental Experimental Procedures. Data have been deposited to the Gene Expression Omnibus, accession number [GEO:XXXXXX].

Supplementary Material

01

Highlights.

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 me3 in immunoblots of extracts masks gene-specific defects.

H3K4me3 at SAGA-dependent genes turns over much faster than at TFIIDdependent genes.

Acknowledgements

We thank Scott Briggs, Brad Cairns, and Yi Jin for strains and plasmids. This work was supported by NIH grants GM46498 and GM56663 to S.B, and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences and NIH grant GM079205 to O.J.R.

Footnotes

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Supplementary Materials

01
NIHMS569506-supplement-01.pdf (3.5MB, pdf)

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