SUMO functions in constitutive transcription and during activation of inducible genes in yeast

Abstract

Transcription factors represent one of the largest groups of proteins regulated by SUMO (small ubiquitin-like modifier) modification, and their sumoylation is usually associated with transcriptional repression. To investigate whether sumoylation plays a general role in regulating transcription in yeast, we determined the occupancy of sumoylated proteins at a variety of genes by chromatin immunoprecipitation (ChIP) using an antibody that recognizes the yeast SUMO peptide. Surprisingly, we detected sumoylated proteins at all constitutively transcribed genes tested but not at repressed genes. Ubc9, the SUMO conjugation enzyme, was not present on these genes, but its inactivation reduced SUMO at the constitutive promoters and modestly decreased RNA polymerase II levels. In contrast, activation of the inducible GAL1, STL1, and ARG1 genes caused not only a striking accumulation of SUMO at all three promoter regions, but also recruitment of Ubc9, indicating that gene activation involves sumoylation of promoter-bound factors. However, Ubc9 inactivation, while reducing sumoylation at the induced promoters, paradoxically resulted in increased transcription. Providing an explanation for this, the reduced sumoylation impaired the cell's ability to appropriately shut off transcription of the induced ARG1 gene, indicating that SUMO can facilitate transcriptional silencing. Our findings thus establish unexpected roles for sumoylation in both constitutive and activated transcription, and provide a novel mechanism for regulating gene expression.

Figure 1.

SUMO associates with constitutively active genes. (A) Representative ChIP analysis of indicated transcriptionally active and silent gene promoters, and a nontranscribed region of Chromosome VII (Intergenic). Occupancy of SUMO and RNAP II was determined by ChIP performed with an Smt3 antibody (SUMO) or an antibody recognizing RNAP II (antibody 8WG16; RNAP II). Control primers recognizing sequences in an untranscribed region of Chromosome V were included in the PCR reactions (*), and input chromatin was also analyzed by PCR for comparison and quantification (INPUT). (B) Quantification of ChIP analyses as shown in A. Quantification (described in the Materials and Methods) is fold over background; a value of 1 indicates no signal detected above background, as indicated with a heavy bar. (C) Occupancy of 8xHIS-tagged Smt3 at indicated genes or intergenic region in the HIS8-SMT3 strain, determined by ChIP as in A and B. (D) Gene diagrams for PYK1, ADH1, and PMA1 indicating gene length, approximate position of transcriptional start site (bent arrow), and regions amplified by indicated ChIP primers. Lengths of genes and amplification products are to scale with respect to each other. (E) Quantification of detailed ChIP analysis of SUMO occupancy at indicated positions across indicated constitutive genes. (F, left) Cell lysates from UBC9-6HA and a control strain were analyzed by Western blot with anti-HA antibody to confirm tagging of Ubc9. (Right) Occupancy of 6xHA-tagged Ubc9 at promoters of indicated genes in the UBC9-6HA strain, determined by ChIP.

Figure 2.

Reduced sumoylation in ubc9-1 cells correlates with reduced RNAP II occupancy at constitutive genes. (A) Whole-cell yeast extracts were prepared from ubc9-1 cells and isogenic wild-type cells, and were analyzed by Western blot with Smt3 antibody (SUMO) and an antibody recognizing the large subunit of RNAP II (Rpb1; antibody 8WG16) as a loading control. Cells were grown at 28°C, or shifted to 37°C for 30 min prior to analysis as indicated. (B) Spot assay comparing growth of ubc9-1 cells and an isogenic wild-type strain on complete minimal medium at the indicated temperature. Approximately 1.5 × 10⁴ cells were plated in the first spot on the left, with fivefold dilutions in each subsequent spot toward the right. Growth was for 2 d. All ChIP experiments using ubc9-1 and the isogenic control were performed using cells grown at 28°C. (C) Occupancy of SUMO and RNAP II was determined at the indicated positions (see Fig. 1D) across PYK1, ADH1, and PMA1 in ubc9-1 cells (gray bars) and the isogenic wild-type strain (black bars). Statistical analysis (Studetnt's t-test) indicates that SUMO levels and RNAP II levels are significantly different in the wild-type and ubc9-1 data sets (P = 0.004 for SUMO, and P = 0.003 for RNAP II). (D) Semiquantitative RT–PCR analysis of steady-state RNA levels of indicated genes in ubc9-1 cells (gray bars) and in isogenic wild-type cells (black bars). RNA levels are not significantly different in the wild-type and ubc9-1 data sets (P > 0.1).

Figure 3.

Activation of inducible genes involves sumoylation of promoter-bound factors. (A) Occupancy of RNAP II, SUMO, and Ubc9-6HA at the inducible GAL1, STL1, and ARG1 gene promoters in normal growth conditions, or in their respective inducing conditions (see the Materials and Methods). (B) Gene diagrams for GAL1 and STL1 as in Figure 1D. (C) Occupancy of RNAP II, SUMO, and Ubc9-6HA at indicated positions across the GAL1 and STL1 genes in uninduced (black bars) or induced conditions (gray bars).

Figure 4.

Sumoylation has a negative effect on transcription of inducible genes. (A, B) Occupancy of SUMO (left) and RNAP II (middle) was determined in uninduced and induced conditions (indicated; as in Fig. 3A) for STL1 (A) and ARG1 (B) in ubc9-1 cells (black bars) and isogenic wild-type cells (gray bars). (Right) Steady-state STL1 (A) and ARG1 (B) mRNA abundance in wild-type and ubc9-1 cells was determined by RT–PCR in untreated or induced cells, as indicated. Values are shown relative to transcript abundance in wild-type cells after induction, which is set to 100. Statistical analysis indicates that RNAP II levels are significantly different in the wild-type and ubc9-1 data sets (P < 0.03 for STL1 data, and P < 0.008 for ARG1 data). RNA levels were also significantly different in wild-type and ubc9-1 cells, with P-values of 0.02 and 0.03 for STL1 and ARG1, respectively. (C) Growth comparison of wild-type and ubc9-1 cells in 0, 0.4, and 0.6 M NaCl in rich medium (left) or minimal medium lacking Val and Ile, and the same medium supplemented with 0.5 μg/mL SM (right). Spot assays were performed as in Figure 2B. Growth was for 2 d at 28°C.

Figure 5.

Sumoylation is required for deactivation of the induced ARG1 gene. (A) ubc9-1 and isogenic wild-type cells transformed with pGcn4-Flag plasmid were grown at 28°C, and cell lysates were prepared from aliquots removed prior to induction (Uninduced), 25 min after adding SM, then 5 min after adding stop mix consisting of fivefold concentrated Val and Ile. Western blot is shown of Gcn4-Flag expression using a Flag antibody and 8WG16 antibody for loading control (Rpb1). (B) Occupancy of SUMO and RNAP II at the ARG1 promoter (position A in Fig. 4B) was determined 25 min after induction with SM, and 5 min after adding the stop mix to wild-type (black bars) and ubc9-1 (gray bars) cells. Statistical analysis of RNAP II levels associated with ARG1 after addition of the stop mix indicates that significant levels were detected in ubc9-1 cells, and no significant levels were detected in wild-type cells (P < 0.04 and P > 0.1, respectively, compared with null hypothesis of fold occupancy equal to 1). (C, left) Transcript abundance of ARG1 was determined by RT–PCR on total RNA isolated from the indicated samples at 0 and 20 min post-addition of SM, and 20 and 40 min post-addition of the stop mix. RNAP I-transcribed 25S RNA analysis is shown as a control. Quantification of three RT–PCR analyses, showing values for 0, 20 and 40 min post-addition of stop mix, is shown at right, normalized to abundance of ARG1 mRNA 20 min post-addition of SM in wild-type and ubc9-1 cells (0 min post-stop).

Figure 6.

Model for the role of SUMO in active transcription in yeast. (A) At constitutive genes, sumoylation (shown as encircled S) of transcription-related factors (gray) prior to recruitment to the promoter facilitates promoter complex assembly or recruitment of RNAP II (Pol II). (B) For inducible genes, when an activator (Ac) is present at sufficient concentration, it binds to the promoter (step 1), and recruits general transcription factors (gray) as well as Ubc9 (step 2). Note that, as for constitutive genes, some transcription-related factors may be sumoylated prior to promoter recruitment. (Step 3) However, the presence of Ubc9 and detection of SUMO at inducible genes during activation implies that sumoylation of promoter-bound factors is part of the activation process. Although targets of sumoylation have not yet been identified at induced promoters, it is possible that the activator itself is targeted, which facilitates its removal from the promoter, perhaps through SUMO-targeted ubiquitin-mediated degradation by the 26S proteasome (not shown). (Step 4) While RNAP II becomes engaged in transcription, SUMO-mediated loss of the activator (or other transcription factors), leads to deactivation of the promoter. (Step 5) The cleared promoter can now become induced again if sufficient activator is present; otherwise, transcription is shut off. Inability to deactivate the promoter by impairing sumoylation leads to prolonged activation of the induced gene, and reduced ability to shut off transcription.