Sumoylation of Transcription Factor Gcn4 Facilitates Its Srb10-Mediated Clearance from Promoters in Yeast

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RESEARCH COMMUNICATION

pression (Gill 2005; Zhao 2007). This is thought to occur by Sumoylation of transcription recruitment of transcriptional corepressor complexes spe- factor Gcn4 facilitates its cifically to SUMO-modified transcription factors bound to promoters (Gill 2005; Garcia-Dominguez and Reyes 2009). Srb10-mediated clearance However, in a few cases, sumoylation of gene-specific transcription factors is associated with activating tran- from promoters in yeast scription (Lyst and Stancheva 2007; Guo and Sharrocks Emanuel Rosonina, Sarah M. Duncan, 2009 and references therein), indicating that SUMO does 1 not have solely a repressive role in transcription. and James L. Manley Supporting a role for SUMO at transcriptionally active Department of Biological Sciences, Columbia University, New genes, we previously demonstrated that the SUMO- York, New York 10027, USA conjugating enzyme Ubc9 is recruited to promoters of activated genes and promoter-bound factors become sumoylated during activation (Rosonina et al. 2010). How- The small ubiquitin-related modifier (SUMO) is a con- ever, the identity of these proteins and the effect of served factor that post-translationally regulates proteins SUMO modification on their specific function during involved in many cellular processes, including gene activation were not determined. Nonetheless, we found transcription. We previously demonstrated that pro- that reducing overall sumoylation at the promoter of the moter-bound factors become sumoylated during activa- induced ARG1 gene did not affect its activation, but in- tion of inducible genes in yeast, but the identity of these stead resulted in elevated transcription levels and impaired factors, and the role of sumoylation in their function, ability to shut down ARG1 transcription, with prolonged was unknown. Here we show that the transcriptional detection of RNA polymerase II (RNAP II) at the promoter after activation had ceased (Rosonina et al. 2010). activator Gcn4 is sumoylated on two specific lysine This pointed to a possible role for SUMO in facilitating residues and in a manner that depends on its ability to clearance of promoter-bound transcription factors, which bind DNA, indicating that sumoylation occurs after can help shut off transcription or possibly reset promoters Gcn4 binding to target promoters. Importantly, this for further activation. functions to facilitate the subsequent removal of the Here we report that the yeast transcriptional activator activator from these promoters after recruitment of RNA Gcn4 is sumoylated at the promoter of target genes. polymerase II, which can prevent inappropriate tran- Interestingly, this sumoylation, which occurs on two scription of target genes. Furthermore, we show that specific Lys residues, facilitates Gcn4 removal during clearance of sumoylated Gcn4 requires the protein ki- ongoing transcription. We further demonstrate that the nase and Mediator complex subunit Srb10, linking acti- protein kinase and Mediator subunit Srb10 also acts to facilitate clearance of Gcn4 from promoters, and this vator removal with target gene transcription. Our study function is strongly stimulated by Gcn4 sumoylation. demonstrates an unexpected role for protein sumoyla- We show that removal of sumoylated Gcn4 through the tion in the process of transcriptional activation. Srb10 pathway is important for preventing excessive Supplemental material is available for this article. accumulation of RNAP II and elevated transcription levels when Gcn4 sumoylation is blocked and a redun- Received December 1, 2011; revised version accepted Janu- dant, but sumoylation-independent, pathway for removal ary 13, 2012. of Gcn4 is disabled. Together, our data support a model in which SUMO marks promoter-bound Gcn4 for clearance through Srb10 phosphorylation-mediated degradation after RNAP II has engaged in transcription, and Protein sumoylation is emerging as an important mech- this is important to ensure tight control of target gene anism of regulating many cellular processes. Sumoyla- expression. tion involves the covalent post-translational modification of specific Lys residues on target proteins with the SUMO (small ubiquitin-related modifier) polypeptide. Results and Discussion The functional consequences of sumoylation vary, gen- erally resulting from altered protein–protein interac- Chromatin-associated Gcn4 is sumoylated at Lys 50 tions, and regulation by SUMO is widespread. Orthologs and Lys 58 of the SUMO protein are found in all eukaryotes, and sumoylated proteins are involved in a wide range of pro- To identify specifically sumoylated proteins at the ARG1 cesses, including DNA repair, chromosome segregation, and promoter and other genes induced by the transcriptional gene expression (Zhao 2007; Makhnevych et al. 2009). activator Gcn4 during amino acid starvation, we looked for One of the largest groups of SUMO-modified proteins in sumoylated factors in Gcn4-containing complexes isolated both yeast and mammals is transcription factors, whose from induced cells by coimmunoprecipitation (co-IP) with sumoylation is usually associated with transcriptional re- Gcn4 (as a 6xHA fusion protein, Gcn4-HA). Expression of Gcn4 was induced by addition of sulfometuron-methyl (SM) to growth medium, which depletes cellular levels of [Keywords: Gcn4; Srb10; sumoylation; transcription] Val and Ile (Falco and Dumas 1985). Anti-HA immuno- 1Corresponding author. E-mail [email protected]. blotting of the Gcn4-HA immunoprecipitations showed Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.184689.111. multiple modified forms of Gcn4, at least four of which

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Sumoylation of Gcn4 comigrated with sumoylated species detected by anti- was eliminated by deleting 40 C-terminal amino acids SUMO immunoblotting of the immunoprecipitations required for DNA binding (Hope and Struhl 1985), but (Fig. 1A; Supplemental Fig. S1A). These sumoylated spe- not necessary for nuclear import (Fig. 1B; Pries et al. cies were not disrupted in immunoprecipitations per- 2002). Similarly, mutation of a single Asn residue (N235; formed in elevated levels of NaCl (0.5 M), but were, in invariant among bZIP domain-containing proteins) to fact, stabilized (Fig. 1A), indicating that they are not either Lys or Thr, which abolishes DNA binding in vitro derived from interacting proteins, but are very likely (Pu and Struhl 1991), greatly reduced Gcn4 sumoylation sumoylated forms of Gcn4 itself. Our data support pre- (Fig. 1B). Finally, by chromatin fractionation, we found vious proteomic studies that identified Gcn4 as a SUMO that sumoylated Gcn4 is stably associated with chro- substrate (Wohlschlegel et al. 2004; Denison et al. 2005) matin, implying that this modification plays a role in and point to sumoylation as a means of regulating Gcn4 regulating the promoter-associated functions of Gcn4 function. (Supplemental Fig. S1D). Furthermore, essentially all To confirm that Gcn4 is, in fact, sumoylated and to nonsumoylatable Gcn4-mt was also associated with identify possible Gcn4 sumoylation sites, we analyzed chromatin, indicating that sumoylation is not required the effects of a number of Lys-to-Arg mutations in Gcn4 for Gcn4 recruitment to DNA. Together, these data demon sumoylation (Fig. 1B). Double Lys-to-Arg mutation onstrate that sumoylated Gcn4 is associated with chro- of residues K50 and K58, both of which fall within matin, and Gcn4 sumoylation most likely occurs after it consensus sumoylation motifs (Fig. 1C; Denison et al. binds target gene promoters. 2005), completely abolished the SUMO-modified forms of Gcn4-6HA, identifying these residues as the probable Sumoylation of Gcn4 facilitates its clearance sites of sumoylation (Fig. 1B). Although chromatin from promoters after recruitment of RNAP II immunoprecipitation (ChIP) analysis of SUMO occupancy on the ARG1 promoter suggests that multiple Next, to determine the function of Gcn4 sumoylation, we promoter-bound factors become sumoylated during characterized yeast strains expressing nonsumoylatable, activation (Supplemental Fig. S1B,C), our data indicate K50,58R mutant Gcn4 (Gcn4-mt) as the only source of that Gcn4 is sumoylated during activation of its target Gcn4. Yeast expressing Gcn4-mt showed no discernible genes. growth defect, compared with isogenic yeast expressing We next investigated whether Gcn4 is sumoylated wild-type Gcn4 (Gcn4-wt), on medium depleted for specifically when bound to DNA. Gcn4 sumoylation amino acids (Supplemental Fig. S2A), nor did the mutations noticeably affect expression of the Gcn4 target genes ARG1 and CPA2, at least under these conditions (Supplemental Fig. S2B; but see below). However, using ChIP time-course analyses, we detected significantly higher levels of Gcn4-mt than Gcn4-wt on the ARG1 and CPA2 promoters ;10–16 min post-induction (Fig. 2A,B), even though both Gcn4-wt and Gcn4-mt were expressed at equal levels (Fig. 2C). At a later time point (22 min), Gcn4-wt and Gcn4-mt occupancies were no longer statistically different (Fig. 2A,B), probably as activator occupancy of target promoters approached satura- tion, indicating that Gcn4 sumoylation regulates its occupancy levels under conditions in which promoter occupancy is not maximal. At an earlier time point (4 min), Gcn4-wt and Gcn4-mt occupancy levels were also equivalent (Fig. 2A,B), indicating that Gcn4 recruitment was not affected by the mutation, but that, over time, Gcn4-mt accumulated on target promoters to greater levels than did Gcn4-wt. Furthermore, the elevated levels of nonsumoylatable Gcn4 at the ARG1 and CPA2 promoters did not result in increased RNAP II levels (Fig. 2A,B), consistent with the lack of effect on transcript levels and pointing to a possible defect in removal of Gcn4-mt from promoters after RNAP II recruitment. Providing support for the idea that this defect was specifically due to blocked sumoylation, we found that Gcn4 occupancy on target promoters also increased in conditions in which global protein sumoylation was impaired Figure 1. Gcn4 is sumoylated at Lys 50 and Lys 58. (A)HA immunoprecipitations from untreated cells or cells induced for (i.e., in yeast expressing a mutant form of Ubc9) (Supple- expression of Gcn4-HA by addition of SM were analyzed by HA and mental Fig. S2C). Although we cannot entirely rule out the SUMO immunoblots (cf. Supplemental Fig. S1). (Asterisks) Putative possibility that sumoylation affects recruitment of Gcn4 sumoylated Gcn4 isoforms; (open circles) unsumoylated Gcn4 iso- to promoters only after the first few minutes of induction, forms. (B) Analysis of HA immunoprecipitations performed from together these data are highly consistent with sumoyla- induced cells expressing indicated Gcn4-HA mutants or controls, as tion being involved in clearance of Gcn4 from target in A.(DCT40) Gcn4 lacking its 40 C-terminal amino acids; (Ig) immunoglobulin band. (C) Diagram of Gcn4 structure, with prob- promoters after RNAP II recruitment, such that block- able sites of sumoylation indicated (encircled S). (DBD) DNA- ing sumoylation causes prolonged association of Gcn4 binding domain. with promoters.

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Rosonina et al.

Figure 2. Gcn4 sumoylation facilitates its clearance from activated target promoters. (A,B) ChIP analysis of wild-type (wt) or K50,58R/ sumoylation site mutant (mt) Gcn4-Flag and RNAP II occupancy on ARG1 (A)andCPA2 (B) promoters at indicated times after induction. The heavy gridline indicates background level (i.e., onefold over background). P-values (see the Materials and Methods) are indicated in parentheses above paired bars where relevant. (C) Flag and control GAPDH immunoblots of whole-cell extracts from cells induced for expression of Gcn4-Flag-wt or Gcn4-Flag-mt for 10 min. At the exposure shown and with a Flag tag, a single SUMO isoform can be detected in the wild-type sample (;70 kDa). (y) Nonspecific band. (D) Model for regulation of promoter-bound or unbound Gcn4 by CDK (Pho85 or Srb10) phosphorylation-dependent, ubiquitin-mediated degradation. See the text for description. (E) HA immunoblot analysis of extracts from PHO85 or pho85D cells treated with DMSO or MG132, induced for expression of Gcn4-HA-wt or Gcn4-HA-mt, then treated with Stop Mix, as indicated.

Sumoylation of Gcn4 enhances its promoter clearance Fig. S2D) in response to the Stop Mix were observed through Mediator component Srb10 between Gcn4-wt and Gcn4-mt, indicating that Gcn4 sumoylation is not involved in this Pho85-mediated deg- We next wished to investigate how sumoylation might radation pathway. Additionally, since mutation of Lys 50 lead to removal of Gcn4 at target promoters. Two path- and Lys 58 did not affect ubiquitin-dependent proteasome- ways are known to result in Gcn4 degradation through mediated degradation of Gcn4 (Fig. 2E), increased pro- ubiquitin-mediated proteolysis, each involving a different moter occupancy of Gcn4-mt was not caused by blocking cyclin-dependent kinase (CDK): (1) Pho85, which phos- possible ubiquitination of these specific Lys residues phorylates Gcn4 when amino acid levels are restored (which, in any case, do not lie within previously reported (Shemer et al. 2002; Bomeke et al. 2006), thereby marking Gcn4 degradation domains) (Meimoun et al. 2000). it for ubiquitination and subsequent degradation through Next, we determined whether SRB10 deletion affects the 26S proteasome (see Fig. 2D,E; Kornitzer et al. 1994; levels of Gcn4 on the ARG1 and CPA2 promoters during Meimoun et al. 2000); and (2) Srb10, a component of the induction. Upon induction of amino acid starvation, Gcn4 RNAP II Mediator complex that is recruited to activated was expressed at somewhat reduced levels in srb10D cells promoters, including those activated by Gcn4 (Qiu et al. compared with wild-type cells (Fig. 3A). In spite of this, 2004). Like Pho85, Srb10 phosphorylates Gcn4, which time-course ChIP analysis demonstrated that after initial targets it for ubiquitination in assays performed in cell recruitment (4 min), as activation continued, significantly extracts (Chi et al. 2001). However, deletion of PHO85 has more Gcn4 was detected at target promoters in srb10D a greater effect on Gcn4 stabilization than SRB10 deletion, cells (Fig. 3B,C). This was not due to a defect in clearance which has an undetectable effect when Gcn4 is expressed of Gcn4 through the Pho85-mediated pathway as amino at high levels (Meimoun et al. 2000; Chi et al. 2001), acid levels were restored, since addition of the Stop Mix suggesting that Srb10 acts only on a fraction of total Gcn4 effectively cleared Gcn4 in both SRB10 and srb10D (Shemer et al. 2002; Irniger and Braus 2003). Thus, these strains (Supplemental Fig. S3B). Although blocking Gcn4 Gcn4 degradation pathways serve different functions, sumoylation and deletion of SRB10 had a similar effect on either of which might be regulated by sumoylation. Gcn4 promoter retention, cells lacking Srb10 addition- We next examined whether Gcn4 sumoylation might ally showed reduced recruitment of RNAP II to Gcn4 tar- be involved in regulating its clearance from target pro- get promoters (Fig. 3B,C; Supplemental Fig. S3A). This is moters by stimulating its degradation through Pho85 consistent with previous observations (Qiu et al. 2004) and or Srb10. We confirmed that restoring amino acid levels likely reflects the fact that, as a component of the Medi- by addition of concentrated Val and Ile (‘‘Stop Mix’’) ator complex present at many active genes, Srb10 has (Rosonina et al. 2010) caused 26S proteasome-dependent general functions in activation of transcription. Nonethe- (i.e., MG132-sensitive) Gcn4 degradation through Pho85 less, our results establish a specific role for Srb10 in clear- (Fig. 2E). Blocking the proteasome with MG132 likely had ance of promoter-bound Gcn4. little or no effect on Gcn4 levels prior to addition of Our data have demonstrated that both SUMO modifi- the Stop Mix because, within 20 min of induction, Gcn4 cation and Srb10 act in clearing Gcn4 from target pro- expression is expected to continue until amino acid moters. We next examined whether both processes func- levels are restored. No differences in degradation (Fig. 2E) tion in the same pathway. Deletion of SRB10 did or clearance from target gene promoters (Supplemental not prevent Gcn4 sumoylation (Supplemental Fig. S3C),

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Sumoylation of Gcn4

by Pho85, reflecting its prominent, SUMO-independent role in clearing of Gcn4 from cells. To address this, we determined the effects of Gcn4 sumoylation in the absence of the Pho85 pathway. Overall levels of Gcn4 occupancy were reduced in pho85D cells (cf. Figs. 2A,B and 4A), which grow slowly compared with wild type (see Fig. 4C). Nonetheless, blocking Gcn4 sumoylation in pho85D cells (by the K50,58R mutation) caused not only increased promoter occupancy of the activator, but also a corresponding increase in RNAP II accumulation and higher transcription of the ARG1 gene (Fig. 4A,B). Although no significant effect was observed for CPA2 in pho85D cells, whose activation was low compared with ARG1 (see RNAP II occupancy in Figs. 2–4), these results demonstrate that blocking Gcn4 sumoylation can, in fact, affect transcription of at least some target genes, but this effect is normally masked by Pho85, likely through

Figure 3. Srb10 functions in clearance of Gcn4 from target promoters during activation, which is facilitated by Gcn4 sumoylation. (A)Flag and control GAPDH immunoblot analysis of extracts from SRB10 (+)or srb10D (D) cells induced for the indicated durations and after addition of Stop Mix. (B,C) ChIP analysis of Gcn4-Flag or RNAP II occupancy on the ARG1 (B)andCPA2 (C) promoters in SRB10 or srb10D cells after induction for indicated time. (D) ChIP analysis as in B and C, performed in srb10D cells expressing either Gcn4-wt or Gcn4-mt, as indicated. indicating that phosphorylation by Srb10 is not a pre- requisite for Gcn4 sumoylation. However, we detected increased Gcn4 sumoylation in srb10D cells (increase of ;30%) (Supplemental Fig. S3C,D), suggesting an accumulation of sumoylated Gcn4 in the absence of Srb10. This would be expected if Srb10 preferentially targets sumoylated Gcn4 for degradation. In strong support of this notion, Gcn4-wt and Gcn4-mt levels on target gene promoters were equivalent during a time course in srb10D cells (Fig. 3D), indicating that Srb10 is required for the Figure 4. Blocking Gcn4 sumoylation causes increased transcrip- increased clearance of wild-type (sumoylatable) Gcn4 from tion of ARG1 in the absence of Pho85. (A) ChIP analysis of Gcn4- target promoters described above. Extending this result, Flag or RNAP II occupancy on the ARG1 and CPA2 promoters in mutation of all five possible Srb10 phosphorylation sites in pho85D cells 16 min after inducing expression of either Gcn4-wt or Gcn4 (Chi et al. 2001) also caused increased Gcn4 pro- Gcn4-mt, as indicated. (B) RT–PCR analysis of ARG1 and CPA2 moter occupancy (Supplemental Fig. 3SE), providing evi- transcript levels in pho85D cells after induction of either Gcn4-wt or Gcn4-mt expression, as indicated. Values are normalized to the level dence that it is indeed phosphorylation of Gcn4 by Srb10 of the indicated gene transcript for Gcn4-wt-expressing cells. (C) that enhances Gcn4 clearance. Together, our data indicate Comparison of growth for the indicated strains. Strains were spotted that Gcn4 sumoylation facilitates its clearance from target on medium containing the indicated level of SM (13 is 0.5 mg/mL) in promoters through an Srb10-dependent pathway. fivefold dilution series and were grown for the indicated durations. (D) Model indicating the role of Gcn4 sumoylation in its Srb10- mediated clearance from target promoters. (1,2) Promoter-bound Blocking Gcn4 sumoylation causes elevated Gcn4 recruits transcription complexes, including Srb10 as part of transcription of ARG1 in the absence of Pho85 the Mediator, Ubc9, and SUMO, to activated target promoters. Upon transcription initiation, Ubc9 sumoylates Gcn4 (3), marking it for In light of the above, we next considered the possibility clearance through an Srb10 phosphorylation-mediated pathway (4), that the consequences of Gcn4 sumoylation on Srb10- clearing the promoter of Gcn4 and possibly other factors (5), which mediated Gcn4 promoter clearance were being masked allows for reactivation of the promoter (1).

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Rosonina et al.

Pho85-mediated degradation of Gcn4 independent of its Thus, we uncovered novel roles for Srb10 and SUMO in sumoylation status. regulating transcription activation through a highly dy- Previously, we showed that globally reduced sumoyla- namic system of activator recruitment and removal, en- tion caused increased transcription of ARG1 as well as suring tight control of gene expression. a growth defect in cells exposed to amino acid starvation We provided evidence that Srb10 plays a key role in (Rosonina et al. 2010). This is reminiscent of observations resetting promoters during transcription by clearing Gcn4. by others demonstrating that overexpression of Gcn4 in- Srb10 phosphorylates Gcn4 in vivo and stimulates its hibits cell growth, an effect that is exacerbated by deletion ubiquitination in in vitro assays (Chi et al. 2001), in- of PHO85 (Meimoun et al. 2000; Shemer et al. 2002). dicating that Srb10 likely functions through phosphory- Consistent with this, specifically blocking Gcn4 sumoyla- lation-dependent ubiquitin-mediated degradation of Gcn4 tion (i.e., in Gcn4-mt) also resulted in a growth defect in (Fig. 4D). Because both Srb10 and Gcn4 are recruited to pho85D cells under more severe amino acid starvation promoters of target genes during activation (Qiu et al. conditions (103–203 SM) (Fig. 4C, cf. growth in lanes 3 2004), it had been speculated that Srb10 acts specifically and 4). This growth defect is possibly a consequence of on promoter-bound Gcn4 (Chi et al. 2001). Our data now overactivity of Gcn4 (by overexpression or blocking of directly demonstrate that Srb10 indeed controls Gcn4 sumoylation-mediated promoter clearance), resulting in levels on promoters of target genes, thereby linking Gcn4 extended expression of amino acid biosynthesis genes clearance with transcription of target genes. In vitro beyond their need, which is energetically costly (Wagner binding assays showed that Srb10 interacts with the acti- 2005). This growth defect demonstrates a synthetic ge- vation domains of Gcn4, Gal4, and other activators (Ansari netic interaction between Gcn4 sumoylation and PHO85, et al. 2002), indicating that Srb10 has the potential to be and, together with our observations in pho85D cells de- involved in resetting additional inducible genes. In support scribed above, reflects redundant or compensatory path- of this, the yeast Gal4 activator was shown to associate ways for elimination of Gcn4 (see Hartman et al. 2001; dynamically with the GAL1 promoter during activation Boone et al. 2007). This explains the necessity of elimi- (Lipford et al. 2005; Collins et al. 2009), and Srb10 is nating Pho85 in order to observe effects of Gcn4 sumoyla- required for full activation of that gene (Hirst et al. 1999 tion on target gene transcription. Interestingly, we found and references therein). Further supporting a role for that Srb10 is essential for growth under these conditions Srb10 in resetting promoters for continued recruitment (Fig. 4C, lanes 5–8), raising the possibility that the Srb10– of RNAP II, in vitro transcription assays showed that Gcn4 sumoylation pathway that we uncovered can be Srb10 functions in disrupting the preinitiation complex, critical for appropriate transcription of Gcn4 target genes. which is necessary for formation of a scaffold that sustains Together, these data indicate that sumoylation of Gcn4 transcriptional reinitiation (Yudkovsky et al. 2000; Liu enables its efficient clearance from target promoters, et al. 2004). By specifically targeting promoter-bound thereby preventing aberrant and detrimental expression of factors such as Gcn4 after RNAP II has initiated transcrip- target genes, specifically under conditions in which tight tion, we showed that an important, likely general, func- control of expression of these genes is necessary, such as in tion of Srb10 is to regulate promoter activity by facilitating the absence of Pho85. multiple cycles of activation. The experiments described above have provided new SUMO-mediated promoter clearance of transcriptional insights into the mechanism of transcriptional activation regulators is likely to be a general and evolutionarily and identified an unexpected role for protein sumoylation conserved mechanism. For example, sumoylation of pro- in this process. These findings give rise to a model (Fig. 4D) moter-bound PARP-1 has been shown to facilitate its whereby Gcn4 is sumoylated at target gene promoters ubiquitin-mediated clearance from the activated HSP70.1 after RNAP II has been recruited, and this modification promoter in mammalian cells (Martin et al. 2009). A specifically activates Srb10-mediated degradation of the human transcription factor that shares homology with activator. The functional consequence of SUMO-enhanced Gcn4, Atf4, contains a consensus sumoylation motif Gcn4 degradation at promoters, which is apparent when within a region that is highly homologous to the region the dominant mechanism of Gcn4 degradation (Pho85 surrounding Gcn4 K58, indicating that sumoylation might pathway) is blocked, is the prevention of unregulated and play a conserved role in regulating both factors. Consistent elevated transcription of target genes. In Pho85-expressing with our observations with Gcn4, a melanoma- and renal cells, blocking sumoylation causes retention of Gcn4 at carcinoma-associated missense substitution found within promoters through the Srb10-dependent pathway, but the a sumoylation site of transcription factor MITF was re- redundant Pho85-dependent pathway acts to remove Gcn4 cently found to impair MITF sumoylation while increasing before it can affect transcription levels. In the absence of its promoter occupancy and the transcription of many Pho85, nonsumoylatable Gcn4 accumulates on promoters target genes (Bertolotto et al. 2011). Furthermore, an un- to a greater degree compared with wild-type Gcn4, causing identified post-translational modification of promoter- increased RNAP II recruitment and higher levels of bound ERa was suggested to be necessary to signal its transcription, since neither the Srb10 nor Pho85 pathway degradation, allowing cycling of activators on ERa target can remove it efficiently. The rate of removal of an activator promoters (Reid et al. 2003). ERa was later shown to be from target promoters, therefore, is not only linked to the sumoylated, and blocking its sumoylation was found to condition of the cell (e.g., through Pho85 upon amino acid impair ERa-induced transcription (Sentis et al. 2005). It will level restoration), but also tightly coupled to transcription be interesting to determine whether SUMO in fact marks itself through both the Pho85 and SUMO/Srb10-mediated promoter-bound ERa for degradation, demonstrating pathways. This affords the cells tightly controlled mech- that SUMO acts to clear and reset promoters for reactiva- anisms to adjust gene expression levels rapidly in response tion across species. After decades of studying the coordi- to changing growth conditions and connects target gene nated recruitment of transcription factors to activated transcription levels with the abundance of an activator. gene promoters that leads to transcriptional initiation,

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Sumoylation of Gcn4 the dynamics involved in maintaining and terminating Gill G. 2005. Something about SUMO inhibits transcription. Curr Opin transcriptional activation are emerging, and our results Genet Dev 15: 536–541. indicate that protein sumoylation plays a central role. Guo B, Sharrocks AD. 2009. Extracellular signal-regulated kinase mitogen-activated protein kinase signaling initiates a dynamic in- terplay between sumoylation and ubiquitination to regulate the Materials and methods activity of the transcriptional activator PEA3. Mol Cell Biol 29: 3204–3218. The yeast strains used are listed in Supplemental Table S1. Construction Hartman JLt, Garvik B, Hartwell L. 2001. Principles for the buffering of of plasmid pGCN4-Flag was previously reported (Rosonina et al. 2010). genetic variation. Science 291: 1001–1004. Strains expressing Flag-tagged Gcn4 (Gcn4-Flag) were used for ChIP exper- Hirst M, Kobor MS, Kuriakose N, Greenblatt J, Sadowski I. 1999. GAL4 is iments and accompanying control immunoblots, while strains expressing regulated by the RNA polymerase II holoenzyme-associated cyclin- Gcn4-HA were used for detecting modified forms of Gcn4. At long dependent protein kinase SRB10/CDK8. Mol Cell 3: 673–678. exposures, immunoblots of Gcn4-Flag showed the same modified forms Hope IA, Struhl K. 1985. GCN4 protein, synthesized in vitro, binds HIS3 seen with Gcn4-HA (data not shown). Plasmid pGCN4-HA was constructed regulatory sequences: Implications for general control of amino acid by replacing the Flag tag sequence with the 6xHA sequence in pGCN4-Flag, biosynthetic genes in yeast. Cell 43: 177–188. and derived mutants were generated by site-directed mutagenesis. Irniger S, Braus GH. 2003. Controlling transcription by destruction: The For induction of Gcn4 target genes by amino acid starvation, yeast were regulation of yeast Gcn4p stability. Curr Genet 44: 8–18. Kornitzer D, Raboy B, Kulka RG, Fink GR. 1994. Regulated degradation grown in minimal medium lacking Ile and Val to an optical density A260 (O.D.) of 0.6–0.8, then induced for 20 min (unless otherwise specified) with of the transcription factor Gcn4. EMBO J 13: 6021–6030. SM (Sigma) at a final concentration of 0.5 mg/mL. To stop induction, Ile Lipford JR, Smith GT, Chi Y, Deshaies RJ. 2005. A putative stimulatory and Val were added to 10 times their normal concentration (‘‘Stop Mix’’) role for activator turnover in gene expression. Nature 438: 113–116. for 10 min. ChIP, RT–PCR RNA analysis, ChIP and RT–PCR quantifica- Liu Y, Kung C, Fishburn J, Ansari AZ, Shokat KM, Hahn S. 2004. Two tion, and yeast growth spot assays were performed as previously described cyclin-dependent kinases promote RNA polymerase II transcrip- (Rosonina et al. 2010). For protein immunoprecipitations, cells were tion and formation of the scaffold complex. Mol Cell Biol 24: 1721– grown as above and lysed by agitation with glass beads. Washes, lysis, 1735. and immunoprecipitations were performed in the presence of 2.5 mg/mL Liu C, Apodaca J, Davis LE, Rao H. 2007. Proteasome inhibition in wild- N-ethylmaleimide and 150 mM NaCl, except as indicated in Figure 1, type yeast Saccharomyces cerevisiae cells. Biotechniques 42: 158– A and B, where 500 mM NaCl was used. Proteasomal inhibition was 162. performed as previously described (Liu et al. 2007) by addition of MG132 Lyst MJ, Stancheva I. 2007. A role for SUMO modification in transcrip- or mock-treated with DMSO for 45 min prior to induction. Quantitative tional repression and activation. Biochem Soc Trans 35: 1389–1392. immunoblotting was performed using the Odyssey system (Li-Cor). Where Makhnevych T, Sydorskyy Y, Xin X, Srikumar T, Vizeacoumar FJ, Jeram statistical analysis was performed, a two-tailed Student’s t-test was used, SM, Li Z, Bahr S, Andrews BJ, Boone C, et al. 2009. Global map of with P-values indicated in parentheses above paired bars within graphs. SUMO function revealed by protein–protein interaction and genetic Antibodies used for ChIP, immunoprecipitation, and immunoblotting were networks. Mol Cell 33: 124–135. anti-Flag M2 (Sigma), rabbit anti-HA (abm), mouse anti-HA (HA.11; Martin N, Schwamborn K, Schreiber V, Werner A, Guillier C, Zhang XD, Covance), and anti-GAPDH (Sigma). Bischof O, Seeler JS, Dejean A. 2009. PARP-1 transcriptional activity is regulated by sumoylation upon heat shock. EMBO J 28: 3534–3548. Meimoun A, Holtzman T, Weissman Z, McBride HJ, Stillman DJ, Fink Acknowledgments GR, Kornitzer D. 2000. Degradation of the transcription factor Gcn4 requires the kinase Pho85 and the SCF(CDC4) ubiquitin-ligase We thank Alan Hinnebusch for sharing yeast strains. We also thank complex. Mol Biol Cell 11: 915–927. Patricia Richard and Dafne Campigli Di Giammartino for discussions and Pries R, Bomeke K, Irniger S, Grundmann O, Braus GH. 2002. Amino comments on the manuscript. This work was supported by grant GM097174 acid-dependent Gcn4p stability regulation occurs exclusively in the from the National Institutes of Health to J.L.M. yeast nucleus. Eukaryot Cell 1: 663–672. Pu WT, Struhl K. 1991. Highly conserved residues in the bZIP domain of References yeast GCN4 are not essential for DNA binding. Mol Cell Biol 11: 4918–4926. Ansari AZ, Koh SS, Zaman Z, Bongards C, Lehming N, Young RA, Qiu H, Hu C, Yoon S, Natarajan K, Swanson MJ, Hinnebusch AG. 2004. Ptashne M. 2002. Transcriptional activating regions target a cyclin- An array of coactivators is required for optimal recruitment of TATA dependent kinase. Proc Natl Acad Sci 99: 14706–14709. binding protein and RNA polymerase II by promoter-bound Gcn4p. Bertolotto C, Lesueur F, Giuliano S, Strub T, de Lichy M, Bille K, Dessen P, Mol Cell Biol 24: 4104–4117. d’Hayer B, Mohamdi H, Remenieras A et al. 2011. A SUMOylation- Reid G, Hubner MR, Metivier R, Brand H, Denger S, Manu D, Beaudouin defective MITF germline mutation predisposes to melanoma and renal J, Ellenberg J, Gannon F. 2003. Cyclic, proteasome-mediated turnover carcinoma. Nature 480: 94–98. of unliganded and liganded ERa on responsive promoters is an integral Bomeke K, Pries R, Korte V, Scholz E, Herzog B, Schulze F, Braus GH. feature of estrogen signaling. Mol Cell 11: 695–707. 2006. Yeast Gcn4p stabilization is initiated by the dissociation of the Rosonina E, Duncan SM, Manley JL. 2010. SUMO functions in constitu- nuclear Pho85p/Pcl5p complex. Mol Biol Cell 17: 2952–2962. tive transcription and during activation of inducible genes in yeast. Boone C, Bussey H, Andrews BJ. 2007. Exploring genetic interactions and Genes Dev 24: 1242–1252. networks with yeast. Nat Rev Genet 8: 437–449. Sentis S, Le Romancer M, Bianchin C, Rostan MC, Corbo L. 2005. Chi Y, Huddleston MJ, Zhang X, Young RA, Annan RS, Carr SA, Deshaies Sumoylation of the estrogen receptor a hinge region regulates its RJ. 2001. Negative regulation of Gcn4 and Msn2 transcription factors transcriptional activity. Mol Endocrinol 19: 2671–2684. by Srb10 cyclin-dependent kinase. Genes Dev 15: 1078–1092. Shemer R, Meimoun A, Holtzman T, Kornitzer D. 2002. Regulation of Collins GA, Lipford JR, Deshaies RJ, Tansey WP. 2009. Gal4 turnover and the transcription factor Gcn4 by Pho85 cyclin PCL5. Mol Cell Biol 22: transcription activation. Nature 461: E7–E8. doi: 10.1038/nature08406. 5395–5404. Denison C, Rudner AD, Gerber SA, Bakalarski CE, Moazed D, Gygi SP. Wagner A. 2005. Energy constraints on the evolution of gene expression. 2005. A proteomic strategy for gaining insights into protein sumoy- Mol Biol Evol 22: 1365–1374. lation in yeast. Mol Cell Proteomics 4: 246–254. Wohlschlegel JA, Johnson ES, Reed SI, Yates JR III. 2004. Global analysis Falco SC, Dumas KS. 1985. Genetic analysis of mutants of Saccharomyces of protein sumoylation in Saccharomyces cerevisiae. J Biol Chem cerevisiae resistant to the herbicide sulfometuron methyl. Genetics 279: 45662–45668. 109: 21–35. Yudkovsky N, Ranish JA, Hahn S. 2000. A transcription reinitiation Garcia-Dominguez M, Reyes JC. 2009. SUMO association with repressor intermediate that is stabilized by activator. Nature 408: 225–229. complexes, emerging routes for transcriptional control. Biochim Bio- Zhao J. 2007. Sumoylation regulates diverse biological processes. Cell phys Acta 1789: 451–459. Mol Life Sci 64: 3017–3033.

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Sumoylation of transcription factor Gcn4 facilitates its Srb10-mediated clearance from promoters in yeast

Emanuel Rosonina, Sarah M. Duncan and James L. Manley

Genes Dev. 2012, 26: Access the most recent version at doi:10.1101/gad.184689.111

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