SUMOylation of HSF1 orchestrates transcriptional and splicing programs during the proteotoxic stress response

Technical Summary: SUMOylation of HSF1 Orchestrates Transcriptional and Splicing Programs During the Proteotoxic Stress Response

Problem Statement
Proteotoxic stress, caused by factors such as heat shock or oxidative damage, threatens cellular homeostasis by inducing protein misfolding and aggregation. Cells counteract this via the Heat Shock Response (HSR), a multi-layered adaptive program orchestrated by the transcription factor Heat Shock Factor 1 (HSF1). While the activation of HSF1 and the subsequent upregulation of molecular chaperones are well-characterized, the signaling mechanisms that coordinate the attenuation and resolution of this response remain incompletely understood. Specifically, how cells ensure the timely disassembly of stress-induced molecular condensates, such as nuclear stress bodies (nSBs), and the restoration of normal splicing patterns is not fully defined. Previous studies suggest a role for post-translational modifications like ubiquitylation and SUMOylation in stress recovery, but a comprehensive, unbiased system-wide analysis of SUMO signaling in this context has been lacking.

Methodology
The authors employed an integrative approach combining pharmacological inhibition, genetic manipulation, and multi-omics analyses to investigate the role of SUMOylation in the proteotoxic stress response.

• Pharmacological Inhibition: HeLa cells were treated with TAK-981, a highly specific inhibitor of the SUMO E1 activating enzyme, to globally inhibit SUMO conjugation by all three paralogs (SUMO-1, SUMO-2, SUMO-3).
• Stress Models: Cells were subjected to transient heat shock (43°C for 1 hour) followed by a recovery phase (2 hours at 37°C). Additional stressors included proteasome inhibition (MG-132), sodium arsenite, cadmium chloride, and mitochondrial HSP90 inhibition (GTPP).
• Omics Analyses:
  • Quantitative Proteomics: TMT-based mass spectrometry was performed on whole-cell, NP-40 soluble, and NP-40 insoluble fractions to assess protein abundance and solubility changes.
  • Transcriptomics: RNA-seq was conducted on wild-type (WT) and HSF1-knockout (KO) HeLa cells to distinguish HSF1-dependent effects.
  • Proximity Proteomics: UltraID (a proximity-dependent biotinylation tool) was used to map the interactome of HSF1 and nuclear stress bodies (nSBs) under stress and recovery conditions.
  • Chromatin Analysis: CUT&RUN assays were utilized to map HSF1 binding sites genome-wide and specifically at the HSATIII locus.
• Genetic Validation: The study utilized siRNA-mediated depletion of specific SUMO paralogs (SUMO-1 vs. SUMO-2/3) and the generation of SUMO-deficient HSF1 mutants (specifically K298R and E299/300A) in an inducible knockdown-complementation system to pinpoint the specific modification site and its functional consequences.

Key Contributions and Results

1. SUMOylation Attenuates HSF1-Driven Gene Expression:
   Inhibition of SUMO conjugation (SUMOi) or depletion of SUMO-2/3 led to sustained and enhanced expression of canonical heat shock proteins (HSPs), particularly HSPA6, during the recovery phase of heat stress. This effect was strictly HSF1-dependent, as it was absent in HSF1 KO cells. The authors identified K298 within the intrinsically disordered region (IDR) of HSF1 as the major SUMO-2/3 acceptor site. Mutating K298 to Arginine (K298R) or the flanking acidic residues (E299/300A) phenocopied the SUMOi effect, resulting in persistent HSPA6 expression even in the absence of chemical inhibition.

2. Mechanism: Enhanced Recruitment of Transcriptional Co-activators:
   Proximity proteomics revealed that in the absence of HSF1 SUMOylation, HSF1 exhibits a stronger association with the general transcription machinery (TFIIB, TFIIF, TFIIE) and transcriptional co-activators, including histone acetyltransferase (HAT) complexes (TADA3, CRTC2) and SWI/SNF chromatin remodeling complexes (SMARCD3). Consequently, SUMOi-treated cells showed increased HSF1 occupancy at canonical heat shock response elements (HSREs) and transcriptional start sites (TSS) of HSP genes. This suggests that SUMOylation normally acts as a "brake" or rheostat, limiting the engagement of HSF1 with productive transcriptional complexes to prevent overshooting of the stress response.

3. Regulation of Nuclear Stress Bodies (nSBs) and Splicing:
   HSF1 drives the transcription of the long non-coding RNA HSATIII, which serves as a scaffold for the assembly of nuclear stress bodies (nSBs). The study found that SUMOi or the expression of the SUMO-deficient HSF1K298R mutant led to the formation of enlarged, persistent nSBs that failed to disassemble during the recovery phase.

   • Splicing Defects: These persistent nSBs sequestered splicing factors (e.g., SRSF6, SON, PRPF38B) that normally reside in nuclear speckles. This sequestration disrupted the recovery of normal splicing patterns, leading to altered intron retention landscapes. Specifically, a subset of introns associated with detained introns showed increased retention upon SUMOi treatment.
   • Dependency: The formation of these enlarged nSBs was dependent on active HSF1 transcription, as blocking transcription with KRIBB11 or dBET6 abrogated the phenotype.

4. Broad Applicability:
   The exacerbation of the stress response by SUMO inhibition was not limited to heat shock but was observed across diverse proteotoxic insults, including proteasome inhibition and mitochondrial stress. Furthermore, in multiple myeloma cell models, combinatorial targeting of the proteasome and SUMO pathways resulted in synergistic activation of the HSF1 program and reduced cell viability.

Significance
The paper defines the SUMOylation of HSF1 at residue K298 as a central regulatory node essential for the timely resolution of the proteotoxic stress response. The authors propose a model where SUMOylation functions as a molecular timer that:

1. Limits the duration and magnitude of HSF1 transactivation by restricting its interaction with co-activators and the transcriptional machinery.
2. Facilitates the disassembly of HSF1-induced molecular condensates (nSBs) during recovery.
3. Ensures the restoration of normal RNA processing and splicing patterns once stress is relieved.

By linking the attenuation of transcriptional output to the disassembly of phase-separated condensates, the study highlights SUMO signaling as a critical mechanism for resetting cellular homeostasis. The findings suggest that the failure to properly resolve these stress-induced condensates due to impaired SUMOylation can lead to prolonged stress signaling and potential dysregulation of RNA metabolism, underscoring the importance of SUMO signaling in maintaining proteostasis and cellular function.