A default silencing mechanism restrains stress-induced genes in C. elegans
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eLife Assessment
This valuable study identifies a novel regulator of stress-induced gene quiescence in C. elegans: the multi-Zinc-finger protein ZNF-236. The work provides evidence for an active mechanism that maintains the repressed state of inducible genes under basal conditions in the absence of stress. The claims for discovery made in the title and abstract are supported by solid experimental data. However, a deeper investigation into the mechanisms of ZNF-236 action could substantially enhance the manuscript's impact and value.
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Abstract
Inducible gene expression programs require that target genes remain silent until the proper activation signal is received, a hallmark of stress-response pathways. This quiescence is typically assumed to be the default state of stress-inducible genes, maintained without active cellular intervention. Using a forward genetic screen for constitutive activation of inducible heat shock proteins (iHSPs) in C. elegans, we found that the multi-zinc-finger protein ZNF-236 is essential for maintaining iHSP quiescence under normal conditions. Loss of znf-236 causes constitutive iHSP expression throughout the genome, affecting both endogenous iHSP loci and iHSP sequences inserted at dispersed chromosomal sites. However, the effect is also chromosomal context-dependent: robustly heat-responsive iHSP transgenes integrated into the ribosomal DNA locus or extrachromosomal arrays are unaffected by znf-236 loss. This differential responsiveness suggests iHSP induction in znf-236 mutants results from a shift in genome organization, rather than from accumulated denatured proteins or engagement of the canonical heat shock response. Our findings demonstrate the existence of a potent ZNF-236-dependent default silencing mechanism that broadly restrains iHSP genes across the genome and helps ensure appropriate iHSP quiescence even at ectopic chromosomal locations. Contrary to prior assumptions, this suggests that quiescence of stress-inducible genes reflects an actively maintained genomic state rather than merely a passive absence of expression.
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eLife Assessment
This valuable study identifies a novel regulator of stress-induced gene quiescence in C. elegans: the multi-Zinc-finger protein ZNF-236. The work provides evidence for an active mechanism that maintains the repressed state of inducible genes under basal conditions in the absence of stress. The claims for discovery made in the title and abstract are supported by solid experimental data. However, a deeper investigation into the mechanisms of ZNF-236 action could substantially enhance the manuscript's impact and value.
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Reviewer #1 (Public review):
Summary:
The paper by ILBAY et al describes a screen in C. elegans for loss-of-function of factors that are presumed to constitutively downregulate heat shock or stress genes regulated by HSF-1. The hypothesis posits an active mechanism of downregulation of these genes under non-stressed conditions. The screen robustly identified ZNF-236, a multi zinc finger containing protein, whose loss upregulates heat-shock and stress-induced prion-like protein genes, but which does not appear to act in cis at the relevant promoters. The authors speculate that ZNF-236 acts indirectly on chromatin or chromatin domains to repress hs genes under non-stressed conditions.
Strengths:
The screen is clever, well-controlled and quite straightforward. I am convinced that ZNF-236 has something to do with keeping heat shock and …
Reviewer #1 (Public review):
Summary:
The paper by ILBAY et al describes a screen in C. elegans for loss-of-function of factors that are presumed to constitutively downregulate heat shock or stress genes regulated by HSF-1. The hypothesis posits an active mechanism of downregulation of these genes under non-stressed conditions. The screen robustly identified ZNF-236, a multi zinc finger containing protein, whose loss upregulates heat-shock and stress-induced prion-like protein genes, but which does not appear to act in cis at the relevant promoters. The authors speculate that ZNF-236 acts indirectly on chromatin or chromatin domains to repress hs genes under non-stressed conditions.
Strengths:
The screen is clever, well-controlled and quite straightforward. I am convinced that ZNF-236 has something to do with keeping heat shock and other stress transcripts low. The mapping of potential binding sites of ZNF-236 is negative, despite the development of a new method to monitor binding sites. I am not sure whether this assay has a detection/sensitivity threshold limit, as it is not widely used. Up to this point, the data are solid, and the logic is easy to follow.
Weaknesses:
While the primary observations are well-documented, the mode of action of ZNF-236 is inadequately explored. Multi Zn finger proteins often bind RNA (TFIII3A is a classic example), and the following paper addresses multivalent functions of Zn finger proteins in RNA stability and processing: Mol Cell 2024 Oct 3;84(19):3826-3842.e8. doi: 10.1016/j.molcel.2024.08.010.). I see no evidence that would point to a role for ZNF-236 in nuclear organization, yet this is the authors' favorite hypothesis. In my opinion, this proposed mechanism is poorly justified, and certainly should not be posited without first testing whether ZNF-236 acts post-transcriptionally, directly down-regulating the relevant mRNAs in some way. It could regulate RNA stability, splicing, export or translation of the relevant RNAs rather than their transcription rates. This can be tested by monitoring whether ZNF-236 alters run-on transcription rates or not. If nascent RNA synthesis rates are not altered, but rather co- and/or post-transcriptional events, and if ZNF-236 is shown to bind RNA (which is likely), the paper could still postulate that the protein plays a role in downregulating stress and heat shock proteins. However, they could rule out that it acts on the promoter by altering RNA Pol II engagement. Another option that should be tested is that ZNF-236 acts by nucleating an H3K9me domain that might shift the affected genes to the nuclear envelope, sequestering them in a zone of low-level transcription. That is also easily tested by tracking the position of an affected gene in the presence and absence of SNF-236. This latter mechanism is also right in line with known modes of action for Zn finger proteins (in mammals, acting through KAP1 and SETDB1). A role for nucleating H3K9me could be easily tested in worms by screening MET-2 or SET-25 knockouts for heat shock or stress mRNA levels. These data sets are already published.
Without testing these two obvious pathways of action (through RNA or through H3K9me deposition), this paper is too preliminary.
Appraisal:
The authors achieved their initial aim with the screen, and the paper is of interest to the field. However, they do not adequately address the likely modes of action. Indeed, I think their results fail to support the conclusion or speculation that ZNF-236 acts on long-range chromatin organization. No solid evidence is presented to support this claim.
Impact:
If the paper were to address and/or rule out likely modes of action, the paper would be of major value to the field of heat shock and stress mRNA control.
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Reviewer #2 (Public review):
Summary:
This manuscript reports the identification of ZNF-236 as a key regulator that maintains quiescence of heat shock inducible genes in C. elegans. Using a forward genetic screen for constitutive activation of an endogenous hsp-16.41 reporter, the authors show that loss of znf-236 leads to widespread, HSF-1-dependent expression of inducible heat shock proteins (iHSPs) and a subset of prion-like stress-responsive genes, in the absence of proteotoxic stress. Transcriptomic analysis reveals that znf-236 mutants partially overlap with the canonical heat shock response, selectively activating highly inducible iHSPs rather than the full HSR program. iHSP transgenes integrated throughout the genome generally become de-repressed in znf-236 mutants, whereas the same constructs on extrachromosomal arrays or …
Reviewer #2 (Public review):
Summary:
This manuscript reports the identification of ZNF-236 as a key regulator that maintains quiescence of heat shock inducible genes in C. elegans. Using a forward genetic screen for constitutive activation of an endogenous hsp-16.41 reporter, the authors show that loss of znf-236 leads to widespread, HSF-1-dependent expression of inducible heat shock proteins (iHSPs) and a subset of prion-like stress-responsive genes, in the absence of proteotoxic stress. Transcriptomic analysis reveals that znf-236 mutants partially overlap with the canonical heat shock response, selectively activating highly inducible iHSPs rather than the full HSR program. iHSP transgenes integrated throughout the genome generally become de-repressed in znf-236 mutants, whereas the same constructs on extrachromosomal arrays or inserted into the rDNA locus re insensitive to znf-236 loss. Using a newly developed method, Transcription Factor Deaminase Sequencing (TFD-seq), the authors show that ZNF-236 binds sparsely across the genome and does not associate with iHSP promoters, supporting an indirect mode of regulation. Physiologically, znf-236 mutants exhibit increased thermotolerance and maintain iHSP expression during aging.
Strengths:
This is a carefully executed and internally consistent study that identifies a new regulator of stress-induced gene quiescence in C. elegans. The genetics are clean and the phenotypes are robust.
Weaknesses:
The manuscript is largely descriptive. It would be substantially strengthened by deeper mechanistic insight into what ZNF-236 does beyond being required for default silencing.
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Reviewer #3 (Public review):
Summary:
The researchers performed a genetic screen to identify a protein, ZNF-236, which belongs to the zinc finger family, and is required for repression of heat shock inducible genes. The researchers applied a new method to map the binding sites of ZNF-236, and based on the data, suggested that the protein does not repress genes by directly binding to their regulatory regions targeted by HSF1. Insertion of a reporter in multiple genomic regions indicates that repression is not needed in repetitive genomic contexts. Together, this work identifies ZNF-236, a protein that is important to repress heat-shock-responsive genes in the absence of heat shock.
Strengths:
A hit from a productive genetic screen was validated, and followed up by a series of well-designed experiments to characterize how the repression …
Reviewer #3 (Public review):
Summary:
The researchers performed a genetic screen to identify a protein, ZNF-236, which belongs to the zinc finger family, and is required for repression of heat shock inducible genes. The researchers applied a new method to map the binding sites of ZNF-236, and based on the data, suggested that the protein does not repress genes by directly binding to their regulatory regions targeted by HSF1. Insertion of a reporter in multiple genomic regions indicates that repression is not needed in repetitive genomic contexts. Together, this work identifies ZNF-236, a protein that is important to repress heat-shock-responsive genes in the absence of heat shock.
Strengths:
A hit from a productive genetic screen was validated, and followed up by a series of well-designed experiments to characterize how the repression occurs. The evidence that the identified protein is required for the repression of heat shock response genes is strong.
Weaknesses:
The researchers propose and discuss one model of repression based on protein binding data, which depends on a new technique and data that are not fully characterized.
Major Comments:
(1) The phrase "results from a shift in genome organization" in the abstract lacks strong evidence. This interpretation heavily relies on the protein binding technique, using ELT-2 as a positive and an imperfect negative control. If we assume that the binding is a red herring, the interpretation would require some other indirect regulation mechanism. Is it possible that ZNF-236 binds to the RNA of a protein that is required to limit HSF-1 and potentially other transcription factors' activation function? In the extrachromosomal array/rDNA context, perhaps other repressive mechanisms are redundant, and thus active repression by ZNF-236 is not required. This possibility is mentioned in one sentence in the discussion, but most of the other interpretations rely on the ZNF-236 binding data to be correct. Given that there is other evidence for a transcriptional role for ZNF-236, and no negative control (e.g. deletion of the zinc fingers, or a control akin to those done for ChIP-seq (like a null mutant or knockdown), a stronger foundation is needed for the presented model for genome organization.
(2) Continuing along the same line, the study assumes that ZNF-236 function is transcriptional. Is it possible to tag a protein and look at localization? If it is in the nucleus, it could be additional evidence that this is true.
(3) I suggest that the authors analyze the genomic data further. A MEME analysis for ZNF-236 can be done to test if the motif occurrences are enriched at the binding sites. Binding site locations in the genome with respect to genes (exon, intron, promoter, enhancer?) can be analyzed and compared to existing data, such as ATAC-seq. The authors also propose that this protein could be similar to CTCF. There are numerous high-quality and high-resolution Hi-C data in C. elegans larvae, and so the authors can readily compare their binding peak locations to the insulation scores to test their hypothesis.
(4) The researchers suggest that ZNF-236 is important for some genomic context. Based on the transcriptomic data, can they find a clue for what that context may be? Are the ZNF-236 repressed genes enriched for not expressed genes in regions surrounded by highly expressed genes?
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Author response:
We thank the reviewers for their insights and suggestions. We appreciate that the reviewers were engaged by both the observations and their interpretation, and consider their interest in further analysis and clarified discussion to be the best possible compliment to this work.
As noted by the reviewers, the working hypothesis of a nuclear organization role for ZNF-236 is just one model. Clarifying this model and potential alternatives will certainly add to the manuscript and this will be a key part of the revision. Beyond this, several suggested analyses should explore extant models, while providing context for considering alternatives. We look forward to carrying out such analyses as feasible and will report them in the revised manuscript.
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