A forward genetic screen identifies znf-236 as a negative regulator of heat shock gene expression

(A) Schematic of the hsp-16.41 locus tagged with mCherry to generate the heat shock reporter strain PD9295. (B) Rationale behind the forward genetic screen: Genetic factors integrating stress signals into the heat shock response by suppressing HSF-1, either directly (gene-x) or indirectly by preventing proteotoxic stress (gene-y), may normally prevent hsp-16.41 induction. Loss-of-function mutations in such genes are predicted to cause constitutive expression of the mCherry::hsp-16.41 reporter. (C) Representative micrographs showing mCherry::hsp-16.41 expression under normal conditions (0 min heat shock), and after heat shock (HS) at 35°C for 30, 60, or 120 minutes, followed by 5 hours of recovery to allow response maturation. Identical microscopy settings were used for all condition. (D) Gene structure of znf-236, showing exon-intron structure, CRISPR-generated deletion alleles, and the region disrupted by each allele. The R477* allele was identified in the EMS screen. Micrographs show constitutive mCherry::hsp-16.41 induction in each znf-236 mutant in the absence of heat shock. In the cc1120 panel, ‘e’ marks an early embryo lacking induction; bottom right image shows induction in an L2 larva. All images in (C) and (D) were acquired under the same microscopy settings. (E) mCherry::hsp-16.41 expression signal measured by high-throughput imaging of the indicated znf-236 alleles. Under no heat shock, mutant alleles display significant induction of mCherry::hsp-16.41. After a 35 °C for 60 minutes followed by 5 hours recovery, mutant alleles display induction of mCherry mCherry::hsp-16.41 of around 50% for cc1120, 75% for cc10213 and 90% for cc9882. Each group contains at least 60 worms. **** P-val <0.001. (F) mCherry::hsp-16.41 expression in znf-236(cc10213) mutants carrying a partial loss-of-function mutation of hsf-1, showing reduced reporter induction. (G) HSF-1::GFP expression in wild-type and a znf-235(cc10216) worm, showing comparable levels.

Inducible heat shock proteins (iHSPs) and a set of stress-associated prion-like or disordered proteins apparently upregulated in znf-236 mutants

(A) Differential gene expression analysis (DESeq2) comparing L4-stage znf-236(cc1120) mutants to stage-matched wild-type animals, shown as a volcano plot. Upregulated gene classes are labelled. (B) Expression levels (reads per million, RPM) of inducible heat shock proteins (iHSPs) in wild-type and znf-236(cc1120) mutants. (C) Expression levels (RPM) of upregulated prion-like or intrinsically disordered proteins in wild-type and znf-236(cc1120) mutants. (D) Genes previously reported as heat shock (HS) induced (Brunquell et al., 2026, [36]) are highlighted on the volcano plot: black dots indicate genes ranked in the top 10, and lime green dots indicate genes ranked in top 100. The primary overlap between heat shock response and response to znf-236 inactivation consists of iHSPs.

znf-236 mutants exhibit thermotolerance and maintain hsp-16.41 induction during aging

(A) Thermotolerance assay of animals exposed to 34°C for 6 hours and scored 24 hours after. n=6 with sample size of 25 animals. (B) High throughput imaging of animals expressing mCherry::hsp-16.41 exposed to heat shock at 35°C for 1 hour and recovery at 20 °C for 5 hours. Each group contains at least 60 worms. **** P-val <0.001.

Identification of genomic binding sites of ZNF-236 using Transcription Factor Deaminase Sequencing (TFD-seq)

(A) Schematic overview of Transcription Factor Deamination Sequencing (TFD-seq), which uses a deaminase-tagged transcription factor to induce localized mutations at native binding sites. An example result demonstrates mutation accumulation consistent with targeted deamination. (B) Mutation load analysis plot showing concentration of specific genomic mutations resulting from ELT-2::deaminase expression in two biological replicates. (C) Mutation load analysis plot showing concentration of specific genomic mutations resulting from ZNF-236::deaminase expression in two biological replicates. (D-F) Mutation accumulation in promoter regions of known ELT-2 target genes, demonstrating specificity and functional relevance of the ELT-2::deaminase fusion. ELT-2 is known as a GATA factor; and “GATA” motifs were found at the mutation accumulation sites. (G-H) Mutation load plots showing two cases of ZNF-236-specific accumulation of localized mutations and their associated nearby genes. (I) Chromosomal distribution of ZNF-236-specific deaminase signatures. (J) Chromosomal distribution of ELT-2-specific deaminase signatures. (K-L) Mutation load plots at genomic regions encompassing abu-1 and pqn-2 (K) and hsp-16.2 and hsp-16.41 (L), which are upregulated in znf-236 mutants. These regions show little to no consistent ZNF-236-mediated deamination, suggesting indirect regulation.

hsp-16.41 in a non-native chromosomal context retains heat-inducibility but loses responsiveness to znf-236 inactivation

(A) A ∼2kb genomic fragment (termed PCR#99) containing the mCherry::hsp-16.41 reporter and its native flanking sequences was PCR-amplified from the PD9295 reporter strain using a specific primer pair. (B) A stably inherited extrachromosomal array (ccEx10214) was generated by injecting PCR#99 along with a co-injection marker rol-6(su1006) into worms harboring an endogenously tagged gfp::hsp-16.41 locus. (C) After a 30-minute of heat shock followed by 5-hour recovery, mCherry::hsp-16.41 expression was higher in ccEx10214 animals compared to animals carrying the single-copy endogenous reporter, indicating increased heat sensitivity of the multi-copy extrachromosomal array. (D) However, this multi-copy, hyper-heat-sensitive extrachromosomal mCherryy::hsp-16.41 failed to respond to znf-236 inactivation.

Genome-wide and chromosomal context-dependent activation of hsp-16.41 in znf-236 mutants

(A) The mCherry::hsp-16.41 gene locus (Figure 5A) was inserted into various chromosomal locations using the MiniMos transposon-based single-copy insertion method. All resulting insertions were heat-inducible, confirming the functionality of the minimal hsp-16.41 construct. 21 of 23 insertions also showed de-silencing in znf-236 mutants, indicating genome-wide regulatory influence of ZNF-236. The two inserts that failed to respond to znf-236 inactivation were both located within the ribosomal DNA (rDNA) repeat region at the distal end of chromosome I. (B) Representative micrographs showing induction of six (one per chromosome) MiniMos insertions in response to znf-236 inactivation. In all six cases, znf-236 is mutated (the allele cc1147) and the endogenous hsp-16.41 is tagged with gfp (the allele cc10203). (C) Representative micrographs of the two rDNA-associated insertions that were unresponsive to znf-236 inactivation are shown. Notably, the endogenous hsp-16.41 locus (marked with gfp) remains responsive to znf-236 inactivation in these same animals, reinforcing that ZNF-236-mediated silencing is context-dependent—as observed with extrachromosomal DNA.

Observed genetic interactions and outcomes, with a model illustrating a potential role of ZNF-236 in the heat shock response pathway

(A) Under unstressed conditions, ZNF-236 restrains hsp-16.41 expression. In the absence of znf-236, HSF-1-dependent hsp-16.41 expression is observed. znf-236 mutants also exhibit increased stress tolerance, the strength of which correlates with the level of hsp-16.41 expression. (B) Models for ZNF-236 in the heat-shock pathway. ZNF-236 activity may be dampened by heat or other proteotoxic stress (green dotted line, question mark), leading to the expression of inducible heat shock proteins. This modulation could shape the rapidity, magnitude, or duration of the response, in parallel to the canonical unfolded protein/HSF-1 pathway. Alternatively, regulation of znf-236 activity alone, without stress causing protein unfolding, could represent a distinct stress-response branch. The epistasis analysis (Figure 1F) uses a hypomorphic hsf-1 allele, as null alleles are inviable. In worms containing both znf-236 and hsf-1 mutant alleles, mCherry::hsp-16.41 expression is observed in some head tissues. This expression could result from residual HSF-1 activity or from HSF-1-independent activation of hsp-16.41 in znf-236 mutants. Therefore, we cannot rule out an hsf-1-independent regulation of hsp genes by znf-236 (purple dotted, question mark).