Dynamic sumoylation is essential in yeast for suppressing stress-induced gene expression in non-stress conditions

Marjan Moallem; J Bryan McNeil; Benjamin G Bergey; Emanuel Rosonina

doi:10.1093/nar/gkag495

. 2026 May 20;54(10):gkag495. doi: 10.1093/nar/gkag495

Dynamic sumoylation is essential in yeast for suppressing stress-induced gene expression in non-stress conditions

Marjan Moallem ^1,^#, J Bryan McNeil ^2,^#, Benjamin G Bergey ³, Emanuel Rosonina ^4,^✉

PMCID: PMC13187839 PMID: 42165130

Abstract

Environmental stresses such as heat shock trigger widespread changes in gene expression, collectively known as the environmental stress response (ESR), along with a global increase in SUMO conjugation. However, whether elevated sumoylation contributes to establishing the ESR gene expression program remains unclear. Here, we show that mutant yeast with constitutively high sumoylation levels due to a defect in the SUMO protease Ulp1, ulp1-mt, displays stress-like gene expression changes, including upregulation of heat shock-induced genes, in the absence of external stress. The ESR regulators Hog1 and Msn2 are constitutively active in this mutant and drive the observed transcriptional changes. Surprisingly, both heat shock and Ulp1 impairment lead to a reduction in levels of SUMO that are stably associated with gene loci, suggesting that increased sumoylation of tightly-bound chromatin factors does not underlie stress-like transcriptional changes. Intriguingly, mutant yeast with reduced sumoylation due to impairment of sumoylation enzymes also show ESR-like transcription effects, including expression of heat shock genes. These findings indicate that disruption of normal sumoylation/desumoylation cycling, rather than absolute SUMO conjugation levels, triggers an ESR-like transcriptional program. This work reveals a new layer of regulation in stress-responsive gene expression and implicates SUMO homeostasis as key in modulating transcriptional stress signaling.

Graphical Abstract

For image description, please refer to the figure legend and surrounding text.

Introduction

In eukaryotes, hundreds of proteins are subject to sumoylation, a post-translational modification that can impact activity, stability, interactions, and localization of its targets [1–7]. Fractionation experiments reveal that most SUMO-conjugated proteins are chromatin-associated, consistent with the frequent identification of SUMO targets involved in DNA metabolism, RNA synthesis, and RNA processing [8]. Among these are histones, multiple components of the general RNA polymerase II (RNAPII) transcription machinery, including TFIIF and RNAPII itself, as well as the majority of sequence-specific transcription factors [8–11], indicating that sumoylation of gene-bound factors may regulate the expression of numerous protein-coding genes. However, sumoylation can also regulate gene expression by modifying cytoplasmic signaling proteins or by controlling the stability or nuclear access of transcription factors [12–16], indicating that its effects are not limited to gene-bound targets. Indeed, extensive evidence supports the idea that SUMO regulates gene expression through multiple mechanisms, contributing to both activation and repression of transcription in a gene- and context-specific manner [11, 17, 18].

Sumoylation involves the covalent and reversible attachment of the 12-kDa SUMO peptide to target proteins through a three-enzyme cascade that is analogous to ubiquitination. The dimeric E1 enzyme, whose subunits are called Aos1 and Uba2 in budding yeast, activates free SUMO peptides and transfers them to the E2 conjugating enzyme [19]. In all eukaryotes, Ubc9 is the sole E2 enzyme that is responsible for the attachment of SUMO to specific lysine residues within target proteins, a process that is facilitated in vivo by E3 ligases [20]. Desumoylation is accomplished by SUMO proteases, including Ulp1 and Ulp2 in yeast, which have distinct targets and roles [19, 21]. Whereas Ulp2 functions in regulating levels of polysumoylation of its targets, Ulp1, which is situated on the inner surface of the nuclear pore complex, processes the SUMO precursor peptide to its mature form and maintains low levels of conjugation for most SUMO targets under normal (i.e. non-stress) growth conditions [22–25]. Indeed, most SUMO conjugations in yeast last mere seconds as a consequence of the continuous activity of Ulp1, an observation that is consistent with sumoylation being a highly transient modification [24, 26, 27].

While cellular levels of sumoylation are generally low under optimal growth conditions, exposure to heat, osmotic, oxidative, and ethanol stresses leads to a rapid elevation of SUMO conjugation [23, 28–32]. This surge in sumoylation, often referred to as the SUMO stress response, occurs largely through a widespread reduction in SUMO deconjugation that results from stress-mediated regulation of SUMO proteases. During ethanol stress, for example, Ulp1 is re-localized to the nucleolus where it cannot access its targets, while heat shock triggers degradation of Ulp1 in yeast and inactivation of several human SUMO proteases [23, 33, 34]. Yeast mutants that are deficient in SUMO conjugation, and human cells lacking SUMO isoforms SUMO2 and SUMO3, cannot survive heat shock, suggesting that the stress-mediated surge in SUMO conjugation serves an evolutionarily conserved protective role during heat stress [23, 35]. Although the precise role for elevated sumoylation during heat and other stresses remains unclear, some have postulated that elevated sumoylation of chromatin associated factors functions to control gene expression as part of the environmental stress response (ESR) [31, 32, 36].

The ESR is a cellular program that enables yeast cells to adapt to diverse environmental stressors by modulating gene expression through multiple mechanisms [37–40]. Although each stress condition elicits a unique transcriptional response, many stresses induce overlapping sets of genes [38, 39]. Central to this response are master regulatory transcription factors that coordinate the expression changes required for survival. Hsf1 is activated in response to heat shock, glucose starvation, and misfolded proteins, regulating hundreds of downstream genes [41, 42]. Msn2 and Msn4 are similarly activated under a range of stress conditions, including heat shock, osmotic stress, and oxidative stress, and function redundantly to control stress-responsive gene expression [43]. Another key regulator is Hog1, a MAP kinase best known for its role in the osmotic stress response, but also involved in other stress responses, including heat shock [44, 45]. Unlike Hsf1, Msn2, and Msn4, Hog1 is activated during heat shock through an indirect mechanism involving phosphorylation via the cell wall integrity pathway, one of the five MAPK signaling pathways in yeast [46]. Once activated, Hog1 cooperates with other transcription factors, including Msn2 and Msn4, to drive the expression of stress-responsive genes.

Here, we explore whether elevated global sumoylation levels play a role in establishing the transcriptional response to stress. We find that non-stressed mutant yeast that harbor constitutively high SUMO conjugation levels show gene expression changes that resemble those that result from heat shock, including highly elevated expression of some ESR genes. Unexpectedly, strains with constitutively low SUMO conjugation levels also show ESR-like gene expression pattern changes, suggesting that it is the disruption of dynamic sumoylation, not simply elevated or reduced SUMO conjugation levels per se, that triggers an ESR-like transcription response. Finally, we implicate activation of Hog1 and, in particular, Msn2 as key for mediating the ESR-like response in non-stress conditions when sumoylation and desumoylation are disrupted. Together, our findings demonstrate that maintaining cycles of SUMO conjugation and deconjugation is important for suppressing the transcriptional stress response in the absence of stress. These results reveal a broader role for sumoylation in stress biology, showing that beyond acting as a rapid stress-induced modification, appropriately balancing levels of sumoylation and desumoylation is needed to restrain stress-response gene expression in unstressed cells.

Materials and methods

Yeast growth

Yeast strains used in this study are listed in Supplementary Table S1, which includes genotype details of the various background strains employed. Cultures were grown in rich medium (YPD), or synthetic complete medium (SC) where specified, shaking at 30°C until they reached optical densities (ODs) between 0.5 and 0.8 at an absorbance of 595 nm. Biological replicates consisted of independent cultures derived from separate colonies of the same strain. For heat shock, cultures were transferred to an incubator set to 42°C (or 37°C where indicated) for the indicated times. For treatment with bathocuproine (BCP; Sigma; cat. 140 910), a stock solution was prepared by dissolving 70 mg in 100 ml of ethanol, then samples were treated with the indicated final concentrations of BCP or with an equal volume of ethanol for controls. To compare growth of different strains, spot assays were performed as follows. Strains were grown overnight in YPD then diluted to an OD (at 595 nm) of 0.2. Samples were then spotted side-by-side in five-fold serial dilutions on YPD solid-media plates. Plates were incubated at either 30°C or 37°C and images were taken after 1–3 days. To generate growth curves, triplicate cultures were examined using the procedure that we previously described, except the starting culture ODs were 0.05 for this study [23]. Growth curve data values, including standard deviations, can be found in Supplementary Table S10.

Generation of new yeast strains

Previously unreported Kl TRP1 and URA3 knockout strains were generated by transformation with polymerase chain reaction (PCR)-amplified DNA fragments that were incorporated at specific genomic loci through homologous recombination [47]. All strains expressing an HA-tagged protein, specifically a C-terminal 6×-HA epitope tag, were generated by insertion of the 6HA::Kl TRP1 PCR amplicon cassette at the natural locus of the gene in the respective parent strains through homologous recombination as previously described [47]. For generating strains that express an mAID-fused protein, PCR was used to assemble and amplify the 6HA-Kl TRP1/mini degron tag to generate proteins with a C-terminal shortened IAA17 degron (mAID), with sequences available upon request [48]. PCR products included ~50 bp of gene-specific sequences for homologous recombination.

Protein lysates and immunoprecipitation

Cultures of 10 ml were harvested through centrifugation at 3000 × g for 5 min, then pellets were washed with 10 ml of immunoprecipitation (IP) buffer, which consists of 50 mM Tris–HCl (pH 8), 150 mM NaCl, 0.1% nonidet P-40 (NP40), supplemented with a protease inhibitor cocktail (Sigma; cat. P8215), 1 mM phenylmethylsulphonyl fluoride, and 2.5 mg/ml N-ethylmaleimide (NEM; Sigma; cat. E3876). Samples were then resuspended in 500 μl of IP buffer. Lysis and IP were performed as described previously [8, 23]. For IPs, 15 μl of pre-conjugated HA agarose beads (Sigma; cat. A2095) were used. For phosphatase treated samples, IP buffer without EDTA and NEM was used, and lysates were treated by adding 600 units/μl of λ phosphatase (NEB; cat. P0753S) in 1× NEBuffer for PMP (50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij-35, and 1 mM MnCl₂, at pH of 7.5), then incubated for 30 min at 30°C. Samples were then boiled in 2× sodium dodecyl sulfate (SDS) sample buffer for 5 min at 95°C.

Western blots

Prior to western blotting, protein lysates were analyzed by SDS–polyacrylamide gel electrophoresis (PAGE). For Hsf1 blots, 6% Phos-tag gels containing 0.05 mmol/l Phos-tag acrylamide solution (NARD Institute; cat. AAL-107) and 0.1 mmol/l MnCl₂ were used and washed with 1 mmol/l EDTA for 10 min prior to transfer. Wet transfers were performed at 100 V for 1 h using a buffer containing 10% methanol, 50 mM Tris, and 380 mM glycine. Nitrocellulose membranes were used in all cases except for analysis with Hsf1, in which case PVDF was used. Primary antibody incubation was performed overnight using the following dilutions: SUMO (Santa Cruz; cat. sc-28649) 1:1000, GAPDH (Sigma; cat. G9545) 1:3000, HA (Sigma; cat. H6908) 1:1000, Hog1 (Santa Cruz; cat. sc-165978) 1:1000, Hog1p (NEB; 92 155) 1:1000, Hsf1 [49] 1:3000. For Supplementary Fig. S1C, HA antibody C29F4 (NEB/Cell Signalling; cat. 3724) was used at a dilution of 1:500. Chemiluminescence imaging was performed with a MicroChemi Imager (DNR).

RNA analysis

For isolating RNA, 15-ml cultures grown in YPD were harvested through centrifugation at 3000 × g for 4 min, flash-frozen, and stored at −80°C for under two weeks before RNA extraction. then RNA was extracted using a phenol-based freeze–heat method that was previously described [8], with the addition of an extra chloroform wash before ethanol precipitation. RNA was stored at a concentration of 2–4 mg/ml at −80°C immediately after precipitation and used to generate complementary DNA (cDNA) within several days. RNA was diluted to a concentration of 24 ng/μl and 5 μl (120 ng) of this dilution was used to generate cDNA for each sample. Reverse transcription was performed using the Bio-Rad iScript cDNA Synthesis Kit (1708891) according to the manufacturer’s recommendations. cDNA was stored at −20°C for under two weeks before real-time PCR (qPCR) analysis. qPCRs were performed following the revised Minimal Information for Publication of Quantitative Real-Time PCR Experiments guidelines (MIQE 2.0) [50] and as previously described using primer pairs listed in Supplementary Table S2, and a CFX Opus real-time PCR detection system (Bio-Rad) [8, 51]. The averages of three biological trials were presented, and standard deviations are plotted as error bars, with statistical analysis performed as described below. For RNA-seq, samples were further purified using the RNeasy kit (Qiagen) and eluted with 100 μl of nuclease free water. Further details of the RNA-seq procedure and bioinformatics analysis are indicated in Supplementary Table S3, and analysis results are shown in Supplementary Table S4. For heat shock and ubc9-1 comparisons (Supplementary Figs S1D–F), previously published datasets were used with database accessions GSE135568 (GEO database) and PRJEB7579 (ENA database), respectively [52, 53]. Details of the analysis of these datasets are indicated in Supplementary Table S4.

Chromatin immunoprecipitation and chromatin fractionation

Chromatin immunoprecipitation (ChIP) was performed as described previously using 50-ml culture volumes and 10 μl of the SUMO antibody for IP [51]. Isolated DNA was eluted in 100 μl of TE buffer and stored at −20°C prior to quantification and analysis by qPCR was performed as previously described using primers listed in Supplementary Table S2 and the CFX Opus real-time PCR detection system (Bio-Rad) [51]. Gene-specific signals are shown relative to the signal at a non-transcribed region of Chromosome V (Fig. 6C) or to input signals (Supplementary Fig. S5B). The averages of three biological trials were presented, and standard deviations are plotted as error bars, with statistical analysis performed as described below. ChIP-seq was performed as previously described, with details of the analysis indicated in Supplementary Tables S5 and S7, and analysis results shown in Supplementary Tables S6 and S8, respectively [51]. SUMO and RNAPII ChIP-seq analyses in the W303a strain, with and without heat shock, were previously described and corresponding datasets were previously deposited to the Gene Expression Omnibus (GEO) database with accession GSE167428 [8]. ChIP-seq and RNA-seq genome alignment views were generated using the Integrative Genomics Viewer (IGV; desktop version 2.16.2) and the sacCer3 yeast genome assembly. Chromatin fractionation was performed using a previously described procedure [54]. “Low-salt” chromatin was extracted using 150 mM KOAc, whereas salt-stable (“high-salt”) chromatin-associated proteins were extracted using 500 mM KOAc.

Statistical analyses

For the statistical analyses shown in Figs 1A,D, 2A, 3B,D, 4E, and 5C, and Supplementary Figs S2 and S3B, GraphPad Prism (version 10.3.1) was used for ANOVA analyses. In each case, ordinary one-way ANOVA was performed with a Fisher’s least significant difference (LSD) test. Sample pairs showing no significant difference are indicated with ns, whereas asterisks indicate significant difference between indicated pairs as follows: *P < .05, **P < .01, ***P < .001, ^****P < .0001. For the statistical analyses shown in Figs 3F and G, 5A, and 6C, and Supplementary Figs S1G and S5A, two-tailed Student’s t-tests were applied, with P-values <.05 indicated with an asterisk. Output for all statistical analyses is presented in Supplementary Table S9. Data for all graphical figures are presented in Supplementary Table S10.

Results

Gene expression patterns in ulp1-mt and ubc9-1 cells resemble stress-induced changes

Previously, we demonstrated that a mild heat shock of 37°C results in the degradation of Ulp1 and a concomitant increase in SUMO conjugation levels in yeast cells [23]. To determine whether this effect is also observed after a more severe heat shock, cultures growing at the optimal temperature of 30°C were transferred to a 42°C incubator for a range of durations, then lysates were prepared and analyzed by immunoblot. As shown in the SUMO immunoblot in Fig. 1A, overall sumoylation levels increased rapidly during the severe heat shock to a maximal level around 30 min before dropping somewhat by 60 min. In strains of two different genetic backgrounds that express an HA epitope-tagged version of Ulp1 from its natural locus, HA immunoblot showed that a severe heat shock also results in the loss of Ulp1 by around 10 min, with virtually none being detected by 30 min (Fig. 1B). Levels of other sumoylation machinery components, including the E1 subunit Uba2, the E2 enzyme Ubc9, and the SUMO protease Ulp2 did not show a temperature-dependent reduction, implying that the effect is specific to Ulp1 (Fig. 1C). The increase in SUMO conjugation levels after heat shock is reminiscent of increased sumoylation observed in a strain harboring a partially defective and temperature-sensitive mutant form of Ulp1, ulp1-mt (also known as ulp1-I615N; [57]). To compare these directly, cultures of the ulp1-mt and its wild-type parent strain, ULP1, were grown continuously at 30°C or heat shocked at 42°C. Side-by-side SUMO immunoblot analysis showed that, indeed, both untreated ulp1-mt and 12-min heat-shocked wild-type cells show a similar level of elevated sumoylation (Fig. 1D). These data strongly support a role for the loss of Ulp1 activity in elevating cellular levels of sumoylation during both mild and severe heat shocks.

Considering the strong connection between sumoylation and transcriptional regulation, one possibility is that elevated sumoylation during heat shock can itself facilitate the establishment, at least in part, of the ESR gene expression pattern. To investigate this, we sought to determine whether there are similarities in the transcriptomes of untreated ulp1-mt cells, which show constitutively high sumoylation, and heat-shocked wild-type cells, which show transiently increased sumoylation. The transcriptome of the ulp1-mt strain has not been reported, so we isolated RNA from ULP1 and ulp1-mt cultures grown at 30°C and performed RNA-seq (Supplementary Table S3 and Supplementary Fig. S1A). As shown in Fig. 1E and Supplementary Table S4, approximately half of all genes express at significantly different levels in the ulp1-mt and ULP1 strains, with 322 showing at least two-fold higher levels, and 43 showing at least two-fold lower levels, in the ulp1-mt strain. Besides its role in continuously deconjugating SUMO targets, Ulp1 is needed for processing SUMO precursor peptides before they can be conjugated [58]. Although the gene expression changes in the ulp1-mt may result to some degree from a drop in free attachable SUMO, our analyses described below strongly imply that these changes derive from impaired deconjugation of SUMO targets. Intriguingly, the gene with the most significant and dramatic difference in expression is the heat-induced gene HSP12, which is upregulated 37-fold in ulp1-mt cells (Fig. 1E, Supplementary Table S4, and see Supplementary Fig. S1B). Consistent with this, the Hsp12 protein is highly expressed in the ulp1-mt strain, even in non-stress conditions (Supplementary Fig. S1C).

Next, we compared the ulp1-mt transcriptome with that of heat-stressed cells obtained from a previously published study in which a wild-type yeast strain grown at 30°C was transferred to 39°C for 20 min [52]. Specifically, we compared gene expression changes in ulp1-mt cells and in heat-shocked wild-type cells, relative to their respective controls (ULP1 and untreated wild-type cells). As shown in Supplementary Figs S1B and S1D, and Supplementary Tables S4 and S10, gene expression changes in the two strains show a moderate level of similarity, with a Pearson coefficient, r, of 0.47. When considering only genes that show significant changes compared to their respective controls, however, there is a strong correlation (r = 0.78; and see heat map in Supplementary Fig. S1D). Intriguingly, when considering only the direction but not the magnitude of change, 71% of genes that are significantly upregulated in heat-shocked cells are also significantly upregulated in ulp1-mt, while 66% of heat shock-downregulated genes are also downregulated in ulp1-mt. Notably, these similarities were observed even though the ulp1-mt and heat-shock RNA-seq analyses were conducted in different labs, using different background strains and conditions. These data indicate that heat shock and impaired Ulp1 activity, both of which result in elevated levels of SUMO conjugation, have similar effects on the transcriptome.

We reasoned that gene expression changes that result from elevated sumoylation in the ulp1-mt strain may be reversed in a strain harboring constitutively low levels of sumoylation. To test this, we compared our ulp1-mt data with a published RNA-seq analysis performed in a strain expressing a partially defective form of Ubc9, ubc9-1, and its parent strain [53]. Although our ulp1-mt analysis and the ubc9-1 study were conducted with different background strains and conditions, surprisingly, as shown in Supplementary Fig. S1E and Supplementary Tables S4 and S10, changes in gene expression due to Ubc9 impairment largely resemble those that result from impairment of Ulp1. Indeed, most genes showing upregulation in the ubc9-1 strain are also upregulated in ulp1-mt, and most downregulated genes in ubc9-1 are also downregulated in ulp1-mt (Supplementary Fig. S1E heatmap). Consistent with this, gene expression changes in ubc9-1 and ulp1-mt show a comparable degree of similarity to the heat-shock data (compare Supplementary Figs S1E and F). Observing that elevating and reducing global sumoylation levels have similar effects on the transcriptome is unexpected. However, this suggests that modulating SUMO conjugation and deconjugation cycles, which we refer to hereafter as “sumoylation cycling,” rather than the presence or absence of the SUMO modification itself, is key to controlling gene expression patterns. As such, when dynamic sumoylation is disrupted by inhibiting either sumoylation (e.g. in ubc9-1) or desumoylation (e.g. in ulp1-mt), similar effects are seen, and intriguingly, these effects resemble those triggered by heat shock.

Disrupting sumoylation or desumoylation results in expression of stress-induced genes

To validate the RNA-seq comparisons described above and to explore the idea that disrupted sumoylation cycles can lead to an ESR-like gene expression pattern, we examined expression levels of stress-induced genes in strains that display constitutively altered SUMO conjugation levels (Fig. 2A). These include the ulp1-mt and ubc9-1 strains mentioned above and four strains that display varying levels of reduced sumoylation. Like ubc9-1, the ubc9-6 strain harbors a partially inactivating point mutation in Ubc9, while in the UBC9-AA strain, fusion to the rapamycin-induced cytoplasmic-targeting anchor away tag itself reduces Ubc9 activity, even in the absence of rapamycin (Fig. 2B and Supplementary Figs S1G and S2; [23]). Similarly, fusion of either of the SUMO E1 enzyme subunits, Aos1 or Uba2, to the mini auxin-induced degron tag, mAID [48], leads to reduced sumoylation, even in the absence of auxin analogs (strains AOS1.mAID and UBA2.mAID; Fig. 2B and Supplementary Fig. S1G). RNA was isolated from each of the six sumoylation-altered strains and their respective wild-type or parent strains, and RT-qPCR was performed on heat shock-inducible genes (HSP12, HSP26, DDR2, and HSP82), the ribosomal protein gene RPS22B, and the constitutively expressed gene PMA1, either with or without a 5-min heat shock at 42°C.

Consistent with the RNA-seq analyses, HSP12 is highly expressed in unstressed ulp1-mt and ubc9-1 strains, strikingly, at levels that are even higher than when their respective wild-type parent strains were heat shocked (Fig. 2A). Heat shock did not result in a large increase in HSP12 expression in these strains, suggesting that it is already near-maximal. A similar effect was observed in the ubc9-6 and AOS1.mAID strains, whereas the UBC9.AA strain showed a more modest increase and the UBA2.mAID strain showed no significant increase in HSP12 levels in unstressed conditions. Notably, of all the sumoylation-deficient strains tested, UBC9.AA and UBA2.mAID have the smallest defect in sumoylation (i.e. smallest reduction compared to their parent strains; Fig. 2B and Supplementary Fig. S1G). The level of HSP12 expression, therefore, appears to be related to the severity of the sumoylation/desumoylation defect in these strains (compare Fig. 2A and B, and Supplementary Fig. S1G). Analysis of HSP26 and DDR2 also revealed high levels of expression in a manner dependent on the severity of the sumoylation defect, but expression levels of HSP82, RPS22B, and PMA1 were unaffected or barely affected by the defects in these strains (Fig. 2A). These results indicate that a subset of ESR genes is highly expressed in non-stress conditions when sumoylation cycling is disrupted, implying that sumoylation and desumoylation of one or more factors is needed to suppress their expression.

Next, we examined whether disrupted sumoylation cycling and constitutive expression of stress-response genes in these strains impacts cell growth in normal and heat-shock conditions. A temperature of 37°C was used to assess temperature sensitivity in this case since many yeast strains are unable to sustain growth at temperatures greater than 37°C [23]. As previously reported, strains ulp1-mt and ubc9-6 show modest growth defects at 30°C, and like ubc9-1, they are unable to sustain growth at 37°C (Fig. 2C and Supplementary Fig. S3A) [23, 57, 59]. Similarly, AOS1.mAID, which shows high expression levels of HSP12, HSP26, and DDR2, is also temperature sensitive. UBC9.AA, which shows a modest increase in expression of these genes, is partially temperature sensitive, while UBA2.mAID, in which expression of these genes is unaffected, shows no growth defect at the elevated temperature (Fig. 2C and Supplementary Fig. S3A). Notably, none of the sumoylation-altered strains display increased SUMO conjugation levels during heat shock, with most showing reduced sumoylation instead (Supplementary Fig. S2). Taken together, the data in Fig. 2 and Supplementary Figs S1G, S2, and S3A reveal a remarkable correlation between the severity of sumoylation cycling defects, the expression of a subset of stress-induced genes, and temperature sensitivity. Paradoxically, constitutively elevated expression of some stress-response genes appears to relate to increased sensitivity to heat stress.

To continue to explore the relationship between sumoylation cycling defects and expression of stress response genes, we generated a strain that harbors both ubc9-6 and ulp1-mt mutations. Compared to the individual mutations, the ubc9-6 ulp1-mt strain shows an intermediate level of SUMO conjugation that is comparable to wild-type yeast (Fig. 3A and Supplementary Figs S1G and S2). Nonetheless, RT-qPCR analysis showed significantly elevated expression for HSP12, HSP26, and DDR2 without heat shock, as observed in the individual ubc9-6 and ulp1-mt strains (compare Fig. 2A with Fig. 3B). Furthermore, restoring sumoylation levels by combining the mutations did not reverse the temperature-sensitivity defect (Fig. 3C and Supplementary Fig. S3A). This supports the idea that it is defects in sumoylation cycling that impacts expression of stress-response genes and viability at elevated temperatures, and not the level of SUMO conjugation itself. Previously, we demonstrated that treatment of yeast cells with the compound 1,10-phenanthroline results in a dramatic and rapid reduction in SUMO conjugation levels by inhibiting the SUMO E1 enzyme [24]. While investigating whether analogs of this drug also impact sumoylation, we found that exposure of yeast cultures to bathocuproine (BCP) instead causes a transient increase in global sumoylation, peaking at around 10 min before returning to basal levels (Fig. 3D, E, F). Although the mechanism by which BCP produces this effect is not yet known, we took advantage of this response to examine whether a transient elevation of sumoylation influences the expression of stress-induced genes. Indeed, as shown in Fig. 3G, treatment with BCP for 10 min led to a sharp increase in expression of HSP12, HSP26, DDR2, and HSP82. Although we cannot rule out that BCP is able to trigger a stress response by unknown mechanisms, these data are consistent with a strong connection between sumoylation and expression of stress-response genes, where any disruption of SUMO conjugation-deconjugation homeostasis leads to their induction.

Altered sumoylation cycling activates Hog1, resulting in induction of some stress-response genes

To explore mechanisms by which disrupting sumoylation cycling can lead to elevated expression of ESR genes, we examined the status of key regulators of the transcriptional response to environmental stresses. Even under normal growth conditions, Hsf1 is associated with up to ~370 genes, including HSP12, HSP26, and HSP82, but not DDR2, and heat shock triggers its phosphorylation that coincides with activation of its target genes [40, 60–62]. To determine whether disrupted sumoylation leads to constitutive activation of Hsf1, lysates were prepared from untreated or heat-shocked cultures, and Hsf1 immunoblots were performed. As shown in Fig. 4A, heat shock resulted in a notable upward shift in migration for Hsf1 that was not apparent when samples were treated with λ phosphatase, confirming that the shift is indeed due to heat-induced phosphorylation. Migration of Hsf1 in the analysis of untreated ulp1-mt and ubc9-6 cells, however, corresponds to the unphosphorylated form, implying that Hsf1 is not constitutively active, and is therefore not likely responsible for the expression of the subset of ESR genes, in these strains (Fig. 4A and B). Interestingly, treatment of yeast with BCP did result in a migration shift for Hsf1, suggesting that, although the drug impacts sumoylation levels, it may induce expression of ESR response genes through an Hsf1-mediated pathway, unlike in untreated ulp1-mt and ubc9-6 (Fig. 4A and B). This is supported by the observation that BCP robustly induces HSP82, an Hsf1-target gene, whereas this gene is barely impacted in strains with constitutive sumoylation defects (see Figs 3G and 2A).

Next, we investigated whether the mitogen-activated kinase Hog1 could be involved. Hog1 has been largely studied for its key role in osmoregulation, but its signaling affects ~70% of ESR genes, likely through its regulation of multiple transcription factors, including Msn2 and Msn4 [63–66]. Furthermore, phosphorylation activates Hog1 in response to high osmolarity, but also upon exposure to other stresses including heat [63, 67]. Using a phosphorylation-dependent Hog1 antibody, we examined lysates derived from untreated or heat shocked cultures by immunoblot. As shown in Fig. 4A and B, phosphorylated Hog1 indeed appeared after heat shock, supporting previous findings. Intriguingly, however, this analysis shows that Hog1 is substantially phosphorylated in untreated ulp1-mt, ubc9-6, and BCP-treated cells. In untreated ulp1-mt cells, the level of Hog1 phosphorylation appears to be somewhat greater than in heat shocked wild-type cells, but heat shock in ulp1-mt further elevates this. In untreated ubc9-6, however, the level of Hog1 phosphorylation is dramatically higher than in heat shocked wild-type cells, and heat shock does not lead to a further increase (Fig. 4A and B). These data indicate that disrupting sumoylation cycling leads to activation of Hog1, which might at least partly explain the ESR-like gene expression pattern in these conditions.

We wished to further explore the relationship between Hog1 and sumoylation. First, we tested whether Hog1 is needed for the increase in sumoylation that takes place during heat shock or for elevated SUMO conjugation in the ulp1-mt strain. However, deletion of HOG1 did not impact either of these, as shown in the immunoblots in Fig. 4C and D. Because disrupted sumoylation cycling triggers Hog1 phosphorylation, but Hog1 is not needed for modulating sumoylation, we presume that activation of Hog1 takes place downstream of altered sumoylation cycling. This raises the intriguing possibility that Hog1 activation during heat stress is a consequence, at least in part, of the heat-triggered drop in SUMO deconjugation levels. To determine whether Hog1 signaling is needed for mediating the gene expression effects of disrupted sumoylation cycling, RT-qPCR was performed using RNA isolated from the ULP1, ulp1-mt, ULP1 hog1Δ, and ulp1-mt hog1Δ strains. As shown in Fig. 4E, in the ulp1-mt background, the high level of expression of HSP12 and DDR2, but not HSP26, was dramatically reduced in the absence of Hog1. These data strongly suggest that disrupted sumoylation cycling in the ulp1-mt strain leads to constitutive Hog1 activation, which consequently results in elevated expression of some ESR genes. Whereas Hog1 appears to be largely responsible for induction of HSP12 and DDR2, another pathway must be involved in the induction of HSP26 in the ulp1-mt strain.

Constitutive expression of stress response genes in ulp1-mt depends on Msn2

The transcriptional activators Msn2 and Msn4 function through an Hsf1-independent pathway to induce expression of ESR genes in response to a variety of stressors, including heat [38, 40, 68, 69]. Although they have overlapping functions, Msn2 is constitutively expressed while Msn4 expression is stress-induced and dependent on Msn2 activation [38]. To determine whether Msn2 is activated and whether MSN4 is induced in conditions of disrupted sumoylation cycling, we examined the expression levels of MSN4 and PGM2, both of which depend on active Msn2 for their activation [38, 70]. RNA was isolated from ulp1-mt, ubc9-1, ubc9-6, and their respective parental wild-type strains, and from a wild-type strain treated with BCP, and RT-qPCR was performed with primers specific for MSN2, MSN4, and PGM2 (Fig. 5A). MSN2 expression is not affected by changes to sumoylation cycling, consistent with it being constitutively expressed under a variety of tested conditions [38]. However, MSN4 expression is elevated in the ulp1-mt, ubc9-1, and ubc9-6 strains, while PGM2 expression was increased in all conditions in which sumoylation cycling was altered. Since these data suggest that Msn2 activity is sensitive to the status of sumoylation in the cell, we examined whether Msn2, and/or Msn4, could be responsible for the expression of ESR genes in the ulp1-mt strain. Deletion of MSN2, MSN4, or both in the ulp1-mt and its parental wild-type strains did not impact sumoylation levels (Fig. 5B). However, RT-qPCR analysis showed that the high level of expression of HSP12, HSP26, and DDR2 in ulp1-mt is strongly dependent on Msn2, but not Msn4 (Fig. 5C).

Our data indicate that disrupted sumoylation cycling leads to activation of Hog1 and Msn2, and that these factors are required for stress-independent expression of ESR genes in the ulp1-mt strain. Whereas Msn2 is required for elevated expression of all tested ESR genes in this strain, expression of HSP26 relies on Msn2 but not Hog1, suggesting that activation of Msn2 in particular is involved. To test this, we compared our ulp1-mt RNA-seq data with data from a previously published study in which gene expression patterns were determined for a strain that conditionally expresses an active mutant form of Msn2, Msn2A6, by microarray [55]. In that study, 194 genes were induced, and 338 repressed, following induced expression of Msn2A6. As shown in Fig. 5D, these gene expression changes showed strong similarity to changes due to impairment of Ulp1 function (r = 0.71), even though the studies were conducted using different background strains and conditions. In particular, most genes that are upregulated due to activation of Msn2 are also activated in the ulp1-mt strain (heatmap in Fig. 5D). In another study, ChIP-seq was used to identify 193 genes whose promoter regions are bound by Msn2 prior to or after nutritional stress and it was determined whether binding of Msn2 led to induction, repression, or had no significant effect (“neutral”) on expression of these genes during the stress [56]. Figure 5E shows the effects of Ulp1 impairment on expression of each of these Msn2-targeted genes, as determined by our ulp1-mt RNA-seq analysis. Strikingly, genes that were previously shown to be bound and induced by Msn2 mostly show elevated expression in the ulp1-mt strain, whereas genes previously associated with a neutral or repressive effect of Msn2 showed a reduced or more muted level of expression in ulp1-mt. Furthermore, we found that deleting the gene encoding Rim15, an Msn2-activating kinase, resulted in significant reduction in expression of HSP12, HSP26, and DDR2 in unstressed ubc9-6 cells (Supplementary Fig. S3B) [71]. Together, these data strongly support that much of the gene expression pattern that is specific to the ulp1-mt strain is due to activation of Msn2, and that an Msn2-mediated pathway is likely to be involved in the upregulation of ESR genes whenever SUMO cycling is disrupted.

Levels of sumoylation stably associated with chromatin loci are reduced during heat shock and in the ulp1-mt strain

Next, we explored whether changes in the sumoylation of chromatin-associated proteins specifically contribute to establishing the stress gene expression pattern. As we showed previously, chromatin fractionation analysis indicates that most SUMO conjugates associate with chromatin (Supplementary Figs S4A and S4B; “low salt”) [8]. However, these conjugates readily dissociate from chromatin when the fractionation is performed with higher salt concentrations, indicating that the majority of sumoylated proteins are only loosely associated with chromatin (Supplementary Figs S4A and S4B; compare “low salt” and “high salt” fractions). Consistent with this, previous SUMO ChIP-seq analyses identified stably associated sumoylated proteins at only ~600 loci, situated primarily at tRNA and ribosomal protein genes, as well as at the promoters of ~150 non-ribosomal protein-coding genes (“non-RPGs”) (Supplementary Table S5) [8, 53]. According to these studies, sumoylation at these three gene types derives primarily from SUMO-modified RNAPIII, Rap1, an RNAPII GTF components, respectively [8, 53, 72]. As such, whereas the bulk of sumoylated proteins interact transiently and dynamically with chromatin, a small subset remains tightly associated with specific loci, raising the possibility that changes in sumoylation at these sites could influence gene expression [8].

To explore this, we performed SUMO ChIP-seq analyses after heat shock and in the ulp1-mt strain. A 12-min 37°C heat shock was chosen to match the conditions of an RNAPII ChIP-seq analysis that we had previously conducted (see below). As shown in Fig. 6A and Supplementary Fig. S4C–E, heat shock resulted in a widespread decrease in stably-associated SUMO signals across all gene types, including the prominent SUMO peaks associated with promoter regions of protein-coding genes (Supplementary Table S6). Since heat shock leads to an overall increase in SUMO conjugation (e.g. Fig. 1A), the decrease in SUMO levels at these loci is an unexpected result. We then examined how constitutively elevated sumoylation, through impaired Ulp1 activity, impacts the genomic SUMO landscape. Fractionation analysis showed that the increased sumoylation in the ulp1-mt strain derives predominantly from conjugates that are loosely or transiently associated with chromatin (Supplementary Fig. S4B). Strikingly, as in heat shock, when SUMO ChIP-seq was performed in the ulp1-mt and ULP1 parent strains, stably-associated SUMO signals were significantly reduced across the genome, at all gene classes (Supplementary Tables S7 and S8, and Fig. 6B). Similarly, deletion of the gene encoding the SUMO protease Ulp2 results in reduced SUMO signals associated with non-RPGs in ChIP-qPCR analysis (Fig. 6C). These findings suggest that the global increase in SUMO conjugation seen during heat shock and in desumoylation mutants occurs on proteins that dynamically associate with chromatin, while stably-associated chromatin factors paradoxically show reduced sumoylation in these conditions.

Previously, we performed an RNAPII ChIP-seq analysis in cultures that were untreated or heat shocked in the same conditions as the heat-shock SUMO ChIP-seq experiment described above [23]. On comparing the analyses, we found that the drop in sumoylation at non-RPGs occurred regardless of whether RNAPII levels also dropped during heat shock, implying that the reduction in sumoylation is not necessarily transcription-dependent (Fig. 6D). Indeed, although both SUMO and RNAPII levels were reduced at RPGs during heat shock, we observed no apparent correlation between heat-shock mediated changes in RNAPII and SUMO occupancy at SUMO-containing non-RPGs (Supplementary Fig. S5A). Intriguingly, however, only five genes showed a significant heat shock-dependent increase in SUMO occupancy, all of which are heat stress-induced (Fig. 6A and D, Supplementary Fig. S5B, and Supplementary Table S6). The pattern of sumoylation across the genes includes a promoter-associated peak and a more diffuse signal across the gene body (Supplementary Fig. 6D; see HSP82, SSA2, HSP26, and KAR2). Promoter-associated SUMO peaks at non-RPGs can be largely attributed to TFIIF sumoylation, while RNAPII is also a known SUMO target suggesting that it may be the source of the signal detected at gene bodies [8, 9]. However, mutations that impair sumoylation of Tfg1 or Rpb1, which are the SUMO-targeted subunits of TFIIF and RNAPII, respectively, did not impact the induction of heat-shock genes, indicating that sumoylation of these chromatin-associated factors is not involved in the transcriptional response to heat shock (Supplementary Fig. S5C). Furthermore, SUMO occupancy is not elevated at these heat shock genes in ulp1-mt even though their expression is induced in that strain (Fig. 6E). Taken together, these data suggest that expression of ESR genes in the ulp1-mt strain results from disrupted sumoylation cycling of one or more factors that are not stably associated with chromatin.

Discussion

In response to environmental stress, in both yeast and mammalian systems, global sumoylation levels increase rapidly while transcription patterns genome-wide are adjusted to establish a stress-adaptive gene expression profile. Although previous studies have shown connections between these two observations, it is not known whether they are mechanistically linked [23, 29, 73]. Our findings show that disrupting sumoylation cycling, by decreasing levels of SUMO conjugation or deconjugation, leads to the induction of ESR genes and an overall ESR-like gene expression pattern, even under optimal, non-stress conditions. This suggests that dynamic sumoylation equilibrium is required to suppress the ESR transcriptional response in the absence of stress in yeast. An analogous effect has not been reported in mammalian systems. Indeed, treatment of mouse and human tumour cell lines with small-molecule inhibitors of the SUMO E1 enzyme produces only minimal changes in gene expression, and does not generate an ESR-like profile [3, 74]. This is somewhat surprising, given the well-established links between sumoylation and cellular stress responses, as well as the fact that most human sequence-specific transcription factors are SUMO substrates [5, 11, 31, 75]. One possibility is that prolonged disruption of SUMO dynamics, like that which occurs in our SUMO-cycling-defective yeast strains, is required for the ESR transcriptional program to emerge. Alternatively, higher eukaryotes may possess additional buffering mechanisms that function in parallel with SUMO to restrain stress-response gene expression under non-stress conditions.

The idea that sumoylation cycling, rather than merely the presence or absence of the SUMO modification itself, serves cellular functions is not new. An analysis of the role of sumoylation of the RNAPII general transcription factor TFIIF showed that both impairing its sumoylation and expressing a “permanently sumoylated” form, through fusion of the targeted subunit with SUMO, leads to reduced binding of RNAPII at active gene promoters, suggesting that dynamic sumoylation of TFIIF is somehow involved in facilitating transcription [8]. In another study, mutant yeast strains expressing partially defective or null forms of Ubc9, Ulp1, or Ulp2 that consequently lead to correspondingly reduced or elevated SUMO conjugation levels, all show significantly reduced lifespans, implicating sumoylation cycling of one or more factors as important in promoting longevity [76]. These studies, and the transient nature of SUMO modifications, support the importance of sumoylation cycling, but uncovering the specific molecular mechanisms through which this dynamic process contributes to cellular functions remains an important goal.

Toward understanding the mechanisms by which disrupted sumoylation causes an ESR-like gene expression profile, our work implicates Hog1 and Msn2, but not Hsf1. We provide evidence that both Hog1 and Msn2 are activated in ulp1 and ubc9 mutant strains grown in non-stress conditions, and both contribute significantly to the anomalous expression of HSP12 and DDR2 in the ulp1-mt background. The activated Hog1 and Msn2 pathways appear, then, to function coordinately in the expression a subset of ESR genes when sumoylation cycling is disrupted. This is supported by work that showed that Hog1 and Msn2 interact with each other to regulate gene expression and are not entirely independent [63, 66, 77]. Nonetheless, our data suggest that Msn2 plays a more important role in establishing gene expression patterns in the ulp1-mt strain, considering that HSP26 expression in that strain is dependent on Msn2 but not Hog1, and that there are strong similarities between the ulp1-mt regulon and the previously reported Msn2 regulons (Fig. 5) [55, 56]. In any case, we find that neither Hog1 nor Msn2 is required for establishing elevated sumoylation in the ulp1-mt strain, which aligns with the observations that Hog1 is not required for the surges in sumoylation seen after heat shock or osmotic stress (Fig. 4C) [73]. Together, these findings support a model in which Msn2 and Hog1 mediate downstream transcriptional responses to disrupted sumoylation cycling, but do not contribute to the underlying elevation in sumoylation levels.

By this model, then, we conclude that Hog1 and Msn2 activation may be a consequence of disrupted sumoylation cycling of one or more factors that lie upstream of Hog1 and Msn2 signaling. One plausible mechanism is through modulation of the protein kinase A (PKA) pathway, which functions upstream of Msn2 and plays a central role in coordinating regulation of gene expression in response to environmental changes [78, 79]. As part of this pathway, under normal conditions, the Rim15 kinase is kept inactive through PKA-dependent phosphorylation [80, 81], but in response to a variety of signals, Rim15 escapes this inhibition, allowing it to phosphorylate and activate Msn2 [71, 81]. Consistent with this model, we find that deletion of RIM15 partially suppresses ESR-like gene expression in ubc9-6 cells, implicating the PKA-Rim15-Msn2 axis. Moreover, hyperactivation of PKA has been shown to suppress Hog1 phosphorylation, implying that PKA can downregulate Hog1 activity in some cases, which suggests that there is a regulatory link between the PKA and Hog1 pathways [82]. Together, these observations support the idea that proper cycling of sumoylation may be important for maintaining the repressive effects of PKA on both Rim15 and Hog1, such that when cycling is disrupted, they become active and lead to Msn2-mediated ESR-like gene expression. Although there is evidence of crosstalk between the PKA pathway and sumoylation in other species, it is not yet clear how sumoylation levels impact the PKA pathway in yeast [83–85]. To advance this model, future work can aim to characterize the effects of modulated sumoylation on PKA activity and to identify the specific SUMO target proteins involved.

Two unexpected observations were made in this study. First, we found that strains that constitutively express a subset of ESR-induced genes show sensitivity to heat stress in a manner that correlates with the expression level of these genes. Whereas one might expect that expression of stress-specific gene products in non-stress conditions could prime cells for a more robust adaptation to elevated temperatures, this was not observed. Although paradoxical, our results are consistent with the finding that activation of heat shock genes is not always necessary for survival at high temperatures [86, 87]. Future work can examine whether only transient expression of ESR genes after exposure to stress, as opposed to continual expression, is critical for stress adaptation. The second unexpected observation is that elevated SUMO conjugation, whether triggered by elevated temperatures or reduced SUMO protease activity, leads to a significant reduction in levels of SUMO that are stably associated with genomic loci. This suggest that conditions that increase SUMO conjugation levels lead to widespread dissociation of tightly-bound sumoylated proteins from chromatin, or they somehow promote desumoylation of these chromatin-bound targets. The former possibility is strongly supported by previous work that demonstrated that sumoylation of some sequence-specific transcription factors in yeast and human cells stimulates their dissociation from DNA [51, 88, 89]. Determining whether specific DNA-bound, sumoylated proteins are actively desumoylated or dissociate from chromatin during heat shock will help distinguish between these possibilities.

Our findings shed light on how altered sumoylation dynamics can regulate the transcriptional response to stress. Based on our data, we propose that exposure to stress leads to a surge in global sumoylation, triggered by Ulp1 degradation. This, in turn, activates the stress-responsive transcription factors Hog1 and Msn2, leading to changes in transcription that establish an ESR gene expression pattern. Identifying the specific factor or factors whose activity is modulated by disrupted sumoylation cycling will be critical to validating this model. Notably, our SUMO ChIP-seq analysis revealed that many genes with altered expression in the ulp1-mt strain do not exhibit significant changes in SUMO occupancy, and that impairing sumoylation of RNAPII or TFIIF does not affect ESR gene expression in this context. Additionally, we were unable to detect Msn2 sumoylation under non-stress, heat shock, or ulp1-mt strain conditions (Supplementary Fig. S5D). Although sumoylated Hog1 was observed, its sumoylation level is unaffected by heat shock (Supplementary Fig. S5E). These observations suggest that sumoylation of chromatin-associated proteins, Msn2, or Hog1 is not likely to be the regulatory node. Future work can examine whether sumoylation or desumoylation of components of the PKA pathway can trigger the activation of Hog1 and Msn2. This would help elucidate how sumoylation cycling homeostasis functions to suppress expression of ESR genes in non-stress conditions, and how modulated sumoylation can serve as an early regulatory signal that coordinates the transcriptional changes needed to survive stress conditions.

Supplementary Material

gkag495_Supplemental_Files

gkag495_supplemental_files.zip^{(10.9MB, zip)}

Acknowledgements

We thank members of the Rosonina Lab, Mark Bayfield, and Yi Sheng for their insightful discussions. Thanks also to Sabine Rospert (University of Freiburg, Germany) who generously provided the Hsf1 antibody.

Author contributions Marjan Moallem (Conceptualization [equal], Formal analysis [equal], Investigation [equal], Writing – original draft [equal]), J. Bryan McNeil (Conceptualization [equal], Formal analysis [equal], Investigation [equal], Methodology [equal]), Benjamin G. Bergey (Formal analysis [supporting], Investigation [supporting], Methodology [supporting]), and Emanuel Rosonina (Conceptualization [equal], Data curation [lead], Formal analysis [equal], Investigation [equal], Project administration [lead], Resources [lead], Supervision [lead], Writing – original draft [equal], Writing – review & editing [equal])

Contributor Information

Marjan Moallem, Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.

J Bryan McNeil, Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.

Benjamin G Bergey, Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.

Emanuel Rosonina, Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.

Supplementary data

Supplementary data is available at NAR online.

Conflict of interest

None declared.

Funding

This work was supported by grants from the Canadian Institutes of Health Research (CIHR; grant number PJT-178112) and the Natural Sciences and Engineering Research Council of Canada (NSERC; grant number RGPIN-2023-05089) to E.R. Funding to pay the Open Access publication charges for this article was provided by CIHR grant PJT-178112.

Data availability

The data underlying this article are available in the GEO database at https://www.ncbi.nlm.nih.gov/geo/ and can be accessed with accession numbers GSE290842 (RNA-seq with ULP1 and ulp1-mt strains) and GSSE290843 (SUMO ChIP-seq in ULP1 and ulp1-mt strains).

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Data Availability Statement

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PERMALINK

Dynamic sumoylation is essential in yeast for suppressing stress-induced gene expression in non-stress conditions

Marjan Moallem

J Bryan McNeil

Benjamin G Bergey

Emanuel Rosonina

Roles

Abstract

Graphical Abstract

Graphical Abstract.

Introduction

Materials and methods

Yeast growth

Generation of new yeast strains

Protein lysates and immunoprecipitation

Western blots

RNA analysis

Chromatin immunoprecipitation and chromatin fractionation

Figure 6.

Statistical analyses

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Results

Gene expression patterns in ulp1-mt and ubc9-1 cells resemble stress-induced changes

Disrupting sumoylation or desumoylation results in expression of stress-induced genes

Altered sumoylation cycling activates Hog1, resulting in induction of some stress-response genes

Constitutive expression of stress response genes in ulp1-mt depends on Msn2

Levels of sumoylation stably associated with chromatin loci are reduced during heat shock and in the ulp1-mt strain

Discussion

Supplementary Material

Acknowledgements

Contributor Information

Supplementary data

Conflict of interest

Funding

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

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