Gcn4 is sumoylated at Lys 50 and Lys 58. (A) HA immunoprecipitations from untreated cells or cells induced for expression of Gcn4-HA by addition of SM were analyzed by HA and SUMO immunoblots (cf. Supplemental Fig. S1). (Asterisks) Putative sumoylated Gcn4 isoforms; (open circles) unsumoylated Gcn4 isoforms. (B) Analysis of HA immunoprecipitations performed from induced cells expressing indicated Gcn4-HA mutants or controls, as in A. (ΔCT40) Gcn4 lacking its 40 C-terminal amino acids; (Ig) immunoglobulin band. (C) Diagram of Gcn4 structure, with probable sites of sumoylation indicated (encircled S). (DBD) DNA-binding domain.

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) and CPA2 (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). (†) 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 pho85Δ cells treated with DMSO or MG132, induced for expression of Gcn4-HA-wt or Gcn4-HA-mt, then treated with Stop Mix, as indicated.

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 srb10Δ (Δ) 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) and CPA2 (C) promoters in SRB10 or srb10Δ cells after induction for indicated time. (D) ChIP analysis as in B and C, performed in srb10Δ cells expressing either Gcn4-wt or Gcn4-mt, as indicated.

Blocking Gcn4 sumoylation causes increased transcription of ARG1 in the absence of Pho85. (A) ChIP analysis of Gcn4-Flag or RNAP II occupancy on the ARG1 and CPA2 promoters in pho85Δ cells 16 min after inducing expression of either Gcn4-wt or Gcn4-mt, as indicated. (B) RT–PCR analysis of ARG1 and CPA2 transcript levels in pho85Δ cells after induction of either Gcn4-wt or Gcn4-mt expression, as indicated. Values are normalized to the level of the indicated gene transcript for Gcn4-wt-expressing cells. (C) Comparison of growth for the indicated strains. Strains were spotted on medium containing the indicated level of SM (1× is 0.5 μg/mL) in 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 Gcn4 recruits transcription complexes, including Srb10 as part of the Mediator, Ubc9, and SUMO, to activated target promoters. Upon transcription initiation, Ubc9 sumoylates Gcn4 (3), marking it for clearance through an Srb10 phosphorylation-mediated pathway (4), clearing the promoter of Gcn4 and possibly other factors (5), which allows for reactivation of the promoter (1).