CCR4‐NOT subunit CCF‐1/CNOT7 promotes transcriptional activation to multiple stress responses in Caenorhabditis elegans

Abstract CCR4‐NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4‐NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome‐wide RNAi screen, we identified a subunit of the CCR4‐NOT complex, ccf‐1, as a requirement for the C. elegans transcriptional response to cadmium and acrylamide stress. Using whole‐transcriptome RNA sequencing, we show that the knockdown of ccf‐1 attenuates the activation of a broad range of stress‐protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and xenobiotic detoxification. Consistently, survival assays show that the knockdown of ccf‐1 decreases C. elegans stress resistance and normal lifespan. A yeast 2‐hybrid screen using a CCF‐1 bait identified the homeobox transcription factor PAL‐1 as a physical interactor. Knockdown of pal‐1 inhibits the activation of ccf‐1 dependent stress genes and reduces C. elegans stress resistance. Gene expression analysis reveals that knockdown of ccf‐1 and pal‐1 attenuates the activation of elt‐2 and elt‐3 under stress that encode master transcriptional co‐regulators of stress response in the C. elegans, and that overexpression of ELT‐2 can suppress ccf‐1's requirement for gene transcription in a stress‐dependent manner. Our findings reveal a new role for CCR4‐NOT in the environmental stress response and define its role in stress resistance and longevity in C. elegans.


| INTRODUC TI ON
In response to environmental stress, organisms can mount various adaptive responses to promote survival. At the cellular level, a major form of adaptive response is the transcriptional activation of genes that serve to prevent or repair cellular damage during periods of stress. Examples of such responses include the induction of xenobiotic detoxification and antioxidant genes to alleviate oxidative stress or the activation of chaperone genes functioning in protein folding to maintain proteostasis under heat shock Sarge et al., 1993). In the nematode Caenorhabditis elegans, various transcriptional pathways that regulate environmental stress response have also promoted longevity (Morley & Morimoto, 2004;Tullet et al., 2008). Consistently, impaired stress response contributes to aging in C. elegans and has also been linked to various human diseases (Hekimi et al., 2011;Morley & Morimoto, 2004;Tullet et al., 2008).
As such, uncovering new insights toward understanding molecular mechanisms of stress response pathways are of widespread interest.
Our previous work in C. elegans showed that exposure to the heavy metal cadmium strongly activates a gene named numr-1 (nuclear-localized metal response), which encodes a putative RNA binding protein required for cadmium resistance (Tvermoes et al., 2010;Wu et al., 2019). The mechanism of cadmium-induced numr-1 activation appears to be distinct from the hallmark induction of metallothionein-encoding genes that maintain homeostasis by scavenging and removing xenobiotic metals (Sabolić et al., 2010).
Given that cadmium is known to induce many types of cellular alterations including oxidative stress, DNA damage, and RNA splicing disruption, it is not surprising that multiple stress response pathways are simultaneously activated to promote cellular resistance (Bertin & Averbeck, 2006;Chomyshen et al., 2022). Additionally, cadmium activates the expression of genes encoding various detoxification enzymes involved in glutathione metabolism that is controlled by the SKN-1 (SkiNhead)/Nrf2 transcription factor . A recent study has also implicated the role of the nuclear hormone receptor HIZR-1 (High Zinc activated nuclear Receptor) in regulating the expression of cadmium and zinc-responsive genes in cooperation with the mediator transcriptional complex (Shomer et al., 2019). The involvement of multiple transcription factors in response to metal stress in C. elegans may also reflect the lack of the MTF-1 (Metal responsive Transcription Factor) gene in nematodes that serves as the conserved regulator of metal-induced transcriptional response in fly, fish, and mammals (Günther et al., 2012). While the requirement of multiple transcription factors involved in stress responses is well recognized, less known are co-regulators that assist in these transcriptional activation events.
In this study, we used numr-1 as a stress marker to identify a new role for the CCR4-NOT (Carbon Catabolite Repression 4 -Negative On TATA-less) complex in regulating C. elegans transcriptional response to multiple environmental stress. The CCR4-NOT complex was originally identified as an eukaryotic deadenylase that post-transcriptionally regulates mRNA stability through poly-A tail removal (Collart, 2016;Nousch et al., 2013). Subsequent studies in yeast have also revealed a role for the CCR4-NOT in transcriptional initiation through interactions with RNA polymerase II, implicating a broad regulatory function of this complex in mRNA synthesis and decay (Collart, 2016). A direct role of the CCR4-NOT complex in stress regulation has also been recently reported in yeast (Mulder et al., 2005). Here, we demonstrate that the C. elegans CCR4-NOT deadenylase subunit ccf-1 (CCR4-associated factor) is broadly required for stress-induced transcriptional programming in C. elegans in response to cadmium and acrylamide. Our results show that ccf-1 is required for a normal lifespan along with stress resistance, and its knockdown also shortens the longevity of multiple long-lived mutants. Through the yeast 2-hybrid (Y2H) system, we identified the homeobox PAL-1 (Posterior Alae) transcription factor as a novel physical interactor of CCF-1 and uncovered a previously undescribed role for this protein in the regulation of stress response genes in C. elegans. Overall, this study provides new insights into genetic regulators of transcriptional response and resistance to environmental stress in C. elegans.

| Components of the CCR4-NOT complex are necessary for the cadmium stress response
We previously showed that the cadmium inducible numr-1 gene encodes as a putative RNA binding protein that influences RNA splicing in C. elegans (Wu et al., 2019). To screen for regulators of this stress response, we performed a genome-wide RNAi screen using a numr-1p::GFP transcriptional reporter to identify genes that when knocked down inhibit the induction of numr-1p::GFP after cadmium exposure (Figure 1a,b). We identified 49 genes that when knocked down reduced numr-1p::GFP after cadmium and had minimal effect on C. elegans development. Enrichment analysis of these 49 genes revealed functional annotation to two protein complexes, the CCR4-NOT complex and the Arp2/3 (Actin-related protein) complex ( Figure 1c; Table S1). Fluorescence levels of numr-1p::GFP after cadmium exposure were highly suppressed when four genes encoding subunits of the CCR4-NOT complex and three genes encoding subunits of the Arp2/3 complex were knocked down by RNAi (Figure 1c).
Representative fluorescent micrographs on the effects of ccf-1, ntl-2, arx-3, and arx-5 RNAi on numr-1p::GFP expression in cadmium are shown ( Figure 1d). The CCR4-NOT complex is well characterized for its role as an mRNA deadenylase that facilitates poly-A tail removal, a cellular process that decreases transcript stability that is highly conserved in yeast, worm, and humans (Nousch et al., 2013;Shirai et al., 2014). However, emerging evidence in yeast suggests that the CCR4-NOT complex is also involved in transcriptional regulation, though this molecular mechanism is less understood (Kruk et al., 2011;Mulder et al., 2005). As such, we focused on characterizing the role of the CCR4-NOT complex in the regulation of stressinduced transcription in this study.
To determine if the CCR4-NOT complex is required for the activation of a broad range of cadmium inducible genes, we used RNAi to knock down the ccf-1/CNOT7 gene that encodes the deadenylase subunit of the CCR4-NOT complex (Nousch et al., 2013). Using qPCR, we found that expressions of various classes of cadmiuminduced genes are significantly reduced in worms fed with ccf-1 RNAi as compared to the empty vector (EV) control after cadmium exposure, including those encoding glutathione s-transferase (gst) and heat shock protein (hsp) genes ( Figure 2a). Consistent with ccf-1 functioning as a requirement for the cadmium stress response, the knockdown of ccf-1 reduced C. elegans cadmium resistance; meanwhile, the knockdown of ccf-1 also significantly reduced C. elegans lifespan (Figure 2b). To determine if other subunits of the CCR4-NOT complex are also required for the activation of cadmium-responsive genes (Figure 2c), we knocked down three other genes identified from the genome-wide RNAi screen that encode subunits of the CCR4-NOT complex and performed qPCR analysis. RNAi knockdown of ntl-2 (NOT-like)/CNOT2 suppressed the activation of 9 out of 10 cadmium-induced genes tested, whereas knockdown of ntl-3/CNOT3 and let-711 (LEThal)/CNOT1 suppressed the activation of 2 and 5 out of 10 cadmium-induced genes, respectively ( Figure 2d).
The relative variance in gene expression levels may suggest a differential requirement of each CCR4-NOT subunit in regulating cadmium induced transcriptional response. It is also possible that the difference may be due to varying degrees of RNAi penetrance between the dsRNA clones. Although RNAi depletion of ccf-1, ntl-2, and ntl-3 all led to F 1 embryonic lethality and let-711 RNAi led to P 0 L3/L4 arrest (data not shown). Overall, these data suggest that subunits of the CCR4-NOT complex are required for C. elegans transcriptional response to cadmium stress and that the ccf-1 deadenylase subunit is essential for normal lifespan and cadmium survival.

| ccf-1 is required for the transcriptional response activated by multiple stressors
Given that knockdown of ccf-1 led to a consistent reduction in the expression of select cadmium inducible genes, we next examined the effect of ccf-1 knockdown on the whole transcriptome before and after cadmium exposure using RNA sequencing (Figure 3a; Table S2). Knockdown of ccf-1 under basal conditions led to differential expression of 3901 genes by more than twofold, with the majority of these genes up-regulated ( Figure 3b). Gene up-regulation in response to ccf-1 knockdown in the absence of stress was also observed for select cadmium inducible genes (numr-1, F57B9.3) in our qPCR data (Figure 2a), and is consistent with the known deadenylase function of CCF-1 where its knockdown results in the retention of mRNA poly-A tail that increases transcript stability (Nousch et al., 2013). Enrichment analysis revealed that genes up-regulated F I G U R E 1 Genome-wide RNAi screen reveals a requirement for genes encoding the CCR4-NOT complex for cadmium-induced activation of numr-1p::GFP.  after ccf-1 knockdown cluster to KEGG pathways including ABC transporters, ribosome biogenesis, and lipid metabolism, whereas genes down-regulated by ccf-1 RNAi cluster to the lysosome pathway ( Figure 3b).
Next, we examined the effects of ccf-1 knockdown on genes that are differentially regulated in cadmium by more than twofold.
Of the 1389 cadmium up-regulated genes, 408 were found to be suppressed by >2-fold in ccf-1 knocked-down worms indicating their dependence on ccf-1 for cadmium-induced gene expression ( Figure 3c). The 29.4% (408/1389) reversion effect (>2-fold change) of cadmium-induced genes by ccf-1 RNAi is 6.25-fold greater than the 4.7% reversion effect observed across genes whose expression is unaffected by cadmium ( Figure S1a). Enrichment analysis of the 408 ccf-1-dependent cadmium genes reveals that they cluster to xenobiotic detoxification pathways and metabolic functions including cytochrome P450 metabolism and glutathione activity ( Figure 3d). To determine if the requirement for ccf-1 in buffering transcript levels is specific to stress, we found that 64 out of 1389 cadmium up-regulated genes are also dependent on ccf-1 under control conditions. However, these 64 genes do not enrich detoxification pathways suggesting that ccf-1 is specifically required for xenobiotic gene expression under stress. We next examined the 413 cadmium down-regulated genes and found that 92 were ccf-1 dependent (22.3% reversion, Figure 3e; vs. 10.8% reversion in noncadmium-responsive genes, Figure S1a); these 92 genes enriched to cuticle structure and collagen/extracellular maintenance (Figure 3f; no enrichment toward KEGG pathway was found). Under control conditions, 26 out of 413 cadmium down-regulated genes were ccf-1 dependent, however, these genes do not enrich any specific pathway. Together, these results demonstrate that ccf-1 is an essential factor in regulating a transcriptional response upon cadmium stress exposure.
As ccf-1 is required for the expression of antioxidant genes in response to cadmium, we then examined if ccf-1 is required for mounting a stress response to acrylamide, which is a potent inducer of oxidative stress (Wu et al., 2017). Using qPCR, we found that the knockdown of ccf-1 reduced the activation glutathione related antioxidant genes after acrylamide exposure including gst-12, gst-30, gst-38, and gcs-1 ( Figure 4a). Next, we expanded this analysis to the whole transcriptome by using RNA sequencing and found that the knockdown of ccf-1 showed a 34.7% and 35.0% reversion rate for acrylamide up and down-regulated genes respectively (Figure 4b,c), both are higher than the ccf-1 knockdown reversion rates of 6.3% (>2-fold down-regulated) and 8.1% (>2-fold up-regulated) observed for genes that do not respond to acrylamide ( Figure S1b). Of the 634 acrylamide up-regulated genes, 220 were found to be ccf-1 dependent and they enrich cytochrome P450 and glutathione metabolism pathways (Figure 4d). Under control conditions, 45 out of 634 acrylamide up-regulated genes were found to be ccf-1 dependent, however, these genes do not enrich xenobiotic detoxification pathways. This pattern is similar to that observed under cadmium where In addition to acrylamide, we show that the knockdown of ccf-1 attenuates the activation of gpdh-1p::GFP in response to osmotic stress (Figure S1c), suggesting a broad role for ccf-1 in regulating multiple types of stress responses beyond acrylamide and cadmium.
Overall, these results demonstrate a transcriptome-wide dependency on ccf-1 for the expression of detoxification genes in response to stress, and that these same stress genes generally do not require ccf-1 for their expression under control conditions.

| Stress-resistant long-lived mutants required ccf-1 for longevity
Transcriptomic analysis revealed that ccf-1 is required for the expression of various stress-responsive genes controlled by the transcription factors SKN-1 and HSF-1 that also promote longevity in C. elegans (Baird et al., 2014;Tullet et al., 2008). To test whether ccf-1 is required for SKN-1 and HSF-1 dependent longevity, we knocked down ccf-1 in the skn-1(k1023) gain of function mutant and a full-length hsf-1(FL) overexpression strain (Baird et al., 2014;Tang & Choe, 2015). Knockdown of ccf-1 reduces wildtype lifespan ( Figure 1b) and we found that it completely suppressed the longevity of the skn-1(k1023) mutant but only partially suppressed the longevity of hsf-1(FL) worms ( Figure 4g and Figure S2a). On EV RNAi, hsf-1(FL) extended median lifespan by +35% compared to N2, but this extension is reduced to +16% when fed with ccf-1 RNAi. The skn-1 gene also acts downstream of the insulin signaling pathway to promote the longevity of the insulin receptor daf-2 mutant (Tullet et al., 2008). Compared to wildtype, the daf-2(e1370) mutant was F I G U R E 2 Components of the CCR4-NOT complex regulate the cadmium stress response. (a) Relative expression of cadmium-responsive genes in N2 worms fed with EV or ccf-1 RNAi under basal conditions or after exposure to cadmium. (b) Cadmium survival and lifespan curves of N2 worms fed with EV or ccf-1 RNAi, p < 0.001 as determined by the log-rank test. Results for trial 1 are shown for cadmium survival and trial 2 for lifespan curve, full results, and statistics for all trials are presented in Table S3. (c) Components of the C. elegans CCR4-NOT complex with * indicating genes identified to be required for cadmium-induced numr-1p::GFP from the RNAi screen. (d) Fold change in expression of cadmium-responsive genes in N2 worms fed with EV, ntl-2, ntl-3, or let-711 under basal conditions or after exposure to cadmium. For a and d, bar graphs display means and error bars indicate the standard deviation of N = 4 samples. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to EV (RNAi) + cadmium as determined by two-way ANOVA with Holm-Sidak post hoc tests.
able to extend its lifespan while fed with ccf-1 RNAi to a similar extent as that of EV RNAi ( Figure S2b; median lifespan +133% in EV RNAi vs +119% in ccf-1 RNAi). Overall, these results show that ccf-1 is completely required for the longevity of the skn-1(k1023) mutant, but only partially required for longevity in the hsf-1(FL) and daf-2(e1370) mutant.

F I G U R E 3
Whole-transcriptome sequencing reveals ccf-1 is broadly required for cadmium-induced gene expression. (a) Experimental workflow used for preparing RNA sequencing samples. (b) Pie chart illustrating the number of genes significantly up-regulated or down-regulated in N2 worms by >2-fold after ccf-1 (RNAi) compared to EV. The bar graph illustrates DAVID analysis of KEGG pathways significantly enriched for genes up-regulated (blue) or down-regulated (yellow) after ccf-1 (RNAi) compared to EV in control conditions. Fold enrichment and FDR values are shown for each KEGG pathway. (c) Scatter-plot of 1389 cadmium up-regulated genes (>2-fold) in N2 worms fed with EV compared to their corresponding expression in N2 worms fed with ccf-1 (RNAi). (d) Enriched KEGG pathways and metabolic functions of 408 ccf-1-dependent cadmium up-regulated genes. (e) Scatter-plot of 413 cadmium down-regulated genes (>2-fold) in N2 worms fed with EV compared to their corresponding expression in N2 worms fed with ccf-1 (RNAi). (f) Enriched metabolic functions and cellular compartments of 92 ccf-1-dependent cadmium down-regulated genes. Reversion % in c and e represent % of genes whose expression was altered by >2-fold in the opposing direction by ccf-1 (RNAi) compared to EV.

| CCF-1 localizes to the nucleus and interacts with the PAL-1 transcription factor
To gain insights into how ccf-1 influences stress-responsive genes, we generated a strain of C. elegans expressing an integrated multicopy ccf-1p::CCF-1::GFP transgene to test if CCF-1 protein expression is influenced by stress. CCF-1::GFP is expressed in multiple tissues and intestinal nuclear CCF-1::GFP can be observed in ~50% of the animals under basal conditions ( Figure S3a). Exposure to cadmium or acrylamide did not alter the relative nuclear distribution or total fluorescence of CCF-1::GFP, suggesting that the expression and localization of the CCF-1 protein are not influenced by stress ( Figure S3b-d). However, the nuclear presence of CCF-1 suggests potential alternative functions other than its characterized cytoplasmic deadenylase role.
Given that knockdown of ccf-1 reduced lifespan and stress resistance (Figure 2b), we next tested how these parameters are affected in worms expressing the multicopy CCF-1::GFP transgene where ccf-1 is overexpressed ( Figure S3e). We found that in the CCF-1::GFP strain, there was no extension to normal lifespan and stress resistance compared to the wildtype ( Figure S3f), suggesting that overexpression of ccf-1 alone is not sufficient in enhancing longevity and stress resistance.
Next, we employed the Y2H system using a GAL4-DBD-CCF-1 fusion protein to probe against a mixed-stage C. elegans GAL4-AD cDNA prey library to identify potential CCF-1 binding partners.
The Y2H screen revealed two consistent prey binding partners of CCF-1 that corresponded to clones encoding the CCR4 and PAL-1 proteins (Figure 5a, Figure S4). CCR4 encodes a catalytic subunit of the CCR4-NOT complex and this protein has previously been shown to directly interact with CCF-1 in C. elegans (Nousch et al., 2013). Meanwhile, PAL-1 encodes the orthologue of the human caudal-related homeobox 1 (CDX1) transcription factor with a characterized role in embryonic posterior patterning and male tail development in C. elegans (Edgar et al., 2001).
To test if PAL-1 co-regulates ccf-1 dependent stress genes, we used RNAi to knockdown pal-1 and performed qPCR to quantify its effects on cadmium and acrylamide-induced gene expressions. Knockdown of pal-1 in the N2 wildtype strain did not lead to substantial changes to cadmium and acrylamide-induced gene expression (data not shown), perhaps due to incomplete penetrance of the pal-1 RNAi. Given that a null mutant is not available, we then repeated the experiment using the RNAi-sensitive rrf-3(pk1426) strain and found that pal-1 knockdown broadly reduced the activation of ccf-1 dependent cadmium genes. (Figure 5b).
Knockdown of pal-1 in the rrf-3(pk1426) strain also reduced cadmium survival compared to EV, suggesting a role for pal-1 in stress resistance ( Figure 5c). Similarly, the knockdown of pal-1 in the rrf-3(pk1426) worms treated with acrylamide attenuated the activation of ccf-1 dependent gst genes and reduced acrylamide survival (Figure 5d,e). To determine whether the reduced stress resistance was caused by potential pleiotropic effects stemming from initiating the RNAi knockdown starting at the L1 development stage, we knocked down pal-1 and ccf-1 in 1-day-old adults for 48 hours followed by exposure to cadmium and acrylamide ( Figure S5a).
Knockdown of both pal-1 and ccf-1 during adulthood reduced F I G U R E 4 Depletion of ccf-1 diminishes C. elegans resistance to acrylamide. (a) Relative expression of gst genes and gcs-1 in worms fed with EV or ccf-1 (RNAi) under basal conditions, or after exposure to acrylamide. Bar graphs display means and error bars indicate the standard deviation of N = 4 samples. *p < 0.05 and ***p < 0.001 compared to EV (RNAi) + acrylamide as determined by two-way ANOVA with Holm-Sidak post hoc tests. Scatter-plot of (b) 634 acrylamide up-regulated genes and (c) 217 acrylamide down-regulated genes in N2 worms fed with EV compared to their corresponding gene expression in N2 worms fed with ccf-1 (RNAi). Reversion % in b and c represent % of genes whose expression was altered by >2-fold in the opposing direction by ccf-1 (RNAi) compared to EV. (d) Enriched KEGG pathways of the 220 ccf-1 dependent acrylamide up-regulated genes. (e) Dopaminergic neuron integrity of worms fed with EV or ccf-1 (RNAi) under basal conditions, or treated with acrylamide. Results are combined from three trials of N = 15 worms per condition scored in each trial. Representative fluorescent micrographs of control or acrylamide-treated worms expressing the dat-1p::GFP reporter fed with EV or ccf-1 (RNAi), scale bar is 50 μm. Error bars indicate the standard error of the mean, ***p < 0.001 as determined by the Chi-square test. (f) Acrylamide survival curve of N2 worms fed with EV or ccf-1 (RNAi), p < 0.001 as determined by the log-rank test. Results for trial 2 are shown for acrylamide survival, full results, and statistics for all trials are presented in Table S3. (g) Lifespan curves of N2 and skn-1(k1023) gain of function worms fed with EV or ccf-1 (RNAi). Results for trial 1 are shown with full results and statistics for all trials are presented in Table S3.
F I G U R E 5 PAL-1 interacts with CCF-1 and regulates stress-responsive genes. (a) Y2H interactions between CCF-1 (bait) and three independent clones of PAL-1 (prey) on the control Leu − /Trp − plate, Leu − /Trp − /His − /25 mM 3AT selection plate, and Leu − /Trp − X-gal assay. The X-gal image is converted from grayscale to blue. (b) Relative expression of cadmium response genes under control or cadmium exposure in rrf-3(pk1426) mutant worms fed with EV or pal-1 (RNAi). (c) Cadmium survival of rrf-3(pk1426) worms fed with EV or pal-1 (RNAi). (d) Relative expression of acrylamide response genes under control or acrylamide exposure in rrf-3(pk1426) worms fed with EV or pal-1 (RNAi). (e) Acrylamide survival of rrf-3(pk1426) worms fed with EV or pal-1 (RNAi). Trial 2 is shown for the survival assays in c and e, full results and statistics for all trials are presented in Table S3. (f) Relative gene expression of stress regulating transcription factors in rrf-3(pk1426) worms fed with EV or pal-1 (RNAi). The student's t-test corrected with Holm-Sidak multiple testing was used with *p < 0.05. (g) Relative expression of elt-2 and elt-3 mRNA levels under control, cadmium, or acrylamide exposure in rrf-3(pk1426) worms fed with EV or pal-1 (RNAi). All bar graphs display means and error bars indicate a standard deviation of N = 4 samples. *p < 0.05, **p < 0.01, and ***p < 0.001 as determined by two-way ANOVA with Holm-Sidak post hoc tests in b, d, and g. resistance to cadmium and acrylamide ( Figure S5b,c), suggesting a post-developmental requirement of both genes in mediating stress resistance.
The 53% reduction in elt-2 mRNA under acrylamide condition was not statistically significant (p = 0.13), this is potentially due to the stringency of the two-way ANOVA test used. In support of elt-2 and elt-3 as downstream targets of PAL-1, we found that the PAL-1 DNA binding motif 5′-GYAATWAA-3′ was present within the 5′ upstream region of both the elt-2 and elt-3 sequences ( Figure S6a). We next examined whether ccf-1 knockdown exerted a similar effect on elt-2 and elt-3 expression using our RNA sequencing data.
Unlike pal-1 RNAi, the knockdown of ccf-1 led to an elevated basal level of elt-2 and elt-3 ( Figure S6b,c), this is consistent with ccf-1's deadenylase activity where the loss of its function increases the poly-A tail length and transcript stability (Figure 3b). However, under stress, we found that the knockdown of ccf-1 attenuated the relative-fold activation of elt-2 and elt-3 mRNA compared to EV ( Figure S6d,e). This suggests that ccf-1 is required for the activation of elt-2 and elt-3 in response to cadmium and acrylamide and these effects under stress are consistent with the patterns observed for pal-1 RNAi (Figure 5g).
Overall, these results show that PAL-1 interacts with CCF-1 protein, and that the knockdown of pal-1 reduces C. elegans survival to cadmium and acrylamide stress by potentially attenuating the mRNA expression of the elt-2 and elt-3 transcription factors that are required to mount a transcription response to environmental stress.

| ELT-2 acts downstream of ccf-1 in a stress and gene-specific manner
To further investigate the relationship between ccf-1 and elt-2, we created an integrated ELT-2::GFP strain that stably overexpresses the ELT-2 protein under its native promoter (Figure 6a,b). ELT-2 was chosen as it serves as the primary co-transcriptional regulator in the intestine, which is a major tissue of the C. elegans stress response.
We chose several cadmium and acrylamide-induced genes that were ccf-1 dependent from our RNA sequencing data and measured how these genes are regulated in the ELT-2::GFP strain after ccf-1 RNAi.
Knockdown of ccf-1 in ELT-2::GFP attenuated the expression of most but not all cadmium-responsive genes compared to EV. Notably, overexpression of ELT-2 suppressed ccf-1's requirement for the expression of gst-4, cyp-34A1, cyp-13A8, and hsp-70 under cadmium ( Figure 6c). We then measured the effects of ccf-1 RNAi in ELT-2::GFP under acrylamide, and surprisingly, we found that the knockdown of ccf-1 did not attenuate the expression of any of the seven genes tested ( Figure 6d). These data suggest that elt-2 acts downstream of ccf-1 and that overexpression of ELT-2 was sufficient to bypass the requirement of ccf-1 in the acrylamide stress response. Conversely, given that ELT-2 overexpression was able to bypass the requirement of ccf-1 in some but not all cadmium-inducible genes, it suggests that elt-2 is only partially required for the cadmium stress response that also involves additional factors downstream of ccf-1 (Figure 6e). Relative mRNA expression of elt-2 in the ELT-2::GFP strain. ***p < 0.001 as determined by student's t-test. Relative mRNA expression of ccf-1 dependent (c) cadmium or (d) acrylamide inducible genes as identified from RNA sequencing measured via qPCR in the ELT-2::GFP strain fed with EV or ccf-1 (RNAi) under control or stress conditions. *p < 0.05, **p < 0.01, and ***p < 0.001 as determined by two-way ANOVA with Holm-Sidak post hoc tests. All bar graphs display means and error bars indicate a standard deviation of N = 4 samples. (e) Proposed mechanism of CCF-1 function in C. elegans stress response regulation. The CCR4-NOT complex serves as a deadenylase complex in the cytoplasm. In response to stress, the CCF-1 subunit together with the PAL-1 transcription factor regulates the activation of stress genes through elt-2 under acrylamide and through elt-2 and other yet-to-be-identified factor(s) under cadmium. It remains to be determined if the CCR4-NOT complex as a whole is involved in the activation of stress genes and if factors other than PAL-1 can interact with CCF-1 to regulate this process.

| DISCUSS ION
Transient activation of gene transcription provides a strategy for cells to adapt and respond to stressful conditions based on environmental demand and is critical for organismal survival. Under stress, transcription factors are recruited to gene promoters to initiate transcription based on the distress signal and the response required, and multiple transcription factors often function in parallel for a synergetic response. Here, we show that the C. elegans CCR4-NOT subunit CCF-1 physically interacts with the PAL-1 transcription factor and both are required for the transcriptional activation of multiple classes of stress-protective genes in response to cadmium and acrylamide. We propose that this mechanism of stress-induced gene regulation in part signals through elt-2, which encodes a transcriptional co-regulator with demonstrated roles in multiple C. elegans stress responses . However, given that an epistasis analysis between ccf-1 and pal-1 was not performed due to the lack of a viable loss of function mutant for either gene, it is possible and likely that ccf-1 also functions with other transcription factors not identified in this study for environmental stress-induced transcriptional regulation (Figure 6e).

| CCR4-NOT in stress response and transcription
A role for the CCR4-NOT complex in stress response was initially demonstrated in yeast where CCR4-NOT mutants showed increased sensitivity to replication stress and defects in accumulating RNR (ribonucleotide reductase) genes in response to DNA damage (Mulder et al., 2005). Mechanistically, it was later shown that CCR4-NOT is recruited to the promoter and open reading frames of RNR genes to facilitate transcription initiation and elongation in response to DNA damage (Kruk et al., 2011). Genomewide cross-linking analyses have also shown that CCR4-NOT is predominantly recruited to SAGA-regulated genes in yeast that typically encode highly inducible genes, suggesting a potential role for CCR4-NOT in stress regulation in yeast (Venters et al., 2011). In human cells, CCR4 serves as a coactivator of several nuclear hormone receptors where CCR4 overexpression enhanced the activation of these receptors while CCR4 knockdown reduced receptor activation and decreased the abundance of hormone-responsive target genes (Garapaty et al., 2008). In mice, the CCF-1 homolog CNOT7 directly interacts with retinoid X receptor β to function as a transcriptional co-activator and knockout of Cnot7 impairs retinoic acid induced transcription (Nakamura et al., 2004). We show in this study that multiple subunits of the CCR4-NOT complex positively promote the transcription of stress-inducible genes in C. elegans. In this instance, the CCF-1 subunit of the CCR4-NOT complex interacts physically and functionally with the PAL-1 transcription factor in promoting stress-induced gene expression.
Knockdown of ccf-1 compromises the oxidative stress resistance of C. elegans as evident by reduced survival to cadmium and acrylamide stress along with increased sensitivity to acrylamide-induced dopaminergic neurodegeneration. As ccf-1 and pal-1 knockdown both affected a broad network of stress-responsive gene classes, we propose this interaction likely indirectly promotes stress resistance by activating the expression of elt-2 and elt-3 that encode master transcriptional co-regulators involved in multiple stress responses .
Given that a wide network of different stress-responsive genes is affected by ccf-1 knockdown, an alternative possibility is that the CCR4-NOT complex may regulate stress gene transcription through chromatin remodeling. Dynamic modification to nucleosome organization at promoters is a well-conserved mechanism that governs the temporal expression of stress-responsive genes (De Nadal et al., 2011). A recent study in yeast has shown that loss of caf1 (yeast orthologue of ccf-1) results in an increased transcript abundance and transcriptional efficiency in all heterochromatic regions, supporting a negative role for the CCR4-NOT complex in promoting gene expression via transcriptional silencing to maintain a heterochromatin state (Monteagudo-Mesas et al., 2022). This is in contrast to the finding of this study where we show that depletion of ccf-1 in

| The elt transcriptional network
The GATA transcription factors elt-2 and elt-3 have both been shown to function with transcription factors in the C. elegans intestine and epidermis respectively to regulate the expression of a wide network of stress-responsive genes. Following infection to Pseudomonas aeruginosa, ELT-2 cooperates with the transcription factors SKN-1 and ATF-7 (Activating Transcription Factor) to promote the expression of different classes of immune genes . ELT-2 functions as the predominant regulator of intestinal gene expression, which is the major site of pathogen and xenobiotic stress response in C. elegans. Consistently, direct gene targets of ELT-2 predicted through SAGE analysis include those functioning in metal detoxification, cytochrome P450 metabolism, and glutathione activity (McGhee et al., 2009). Analogous to ELT-2, ELT-3 appears to also function as a stress transcriptional co-regulator with the distinction that ELT-3 primarily acts in the epidermis . In response to oxidative stress, ELT-3 directly binds to the SKN-1 transcription factor to promote the expression of gst-related antioxidant genes (Hu et al., 2017).
ChIP analysis indicates that one-third of ELT-3's targets are also bound directly by the SKN-1 transcription factor, reinforcing the role of ELT-3 as a co-regulator of SKN-1 .
In response to pathogen infection and osmotic stress, ELT-3 controls the expression of neuropeptide-like protein genes potentially through a mechanism involving the STA-2 (Signal Transducer and Activator) transcription factor (Dodd et al., 2018). Lastly, ELT-3 is also required for the expression of DAF-16-regulated genes in the hypodermis, suggesting that it also functions within the insulin/ IGF-1 pathway (Zhang et al., 2013).
We have previously shown that oxidative stress promotes the nuclear localization of SKN-1::GFP in both the intestine and the epidermis, and this is supported by the up-regulation of the gst-4p::GFP reporter in both tissues (Wu et al., 2016). Knockdown of skn-1 in either the intestine or epidermis alone reduces C. elegans resistance to oxidative stress, supporting the idea that SKN-1 activity in both tissues is critical for oxidative stress resistance (Wu et al., 2016). Interestingly, our data show that overexpression of the intestinal transcriptional co-factor ELT-2 can suppress the requirement of ccf-1 in the transcription of acrylamide-responsive genes tested. Given that majority of ccf-1-dependent acrylamideresponsive genes function in xenobiotic detoxification which is known to be controlled by the SKN-1 transcription factor, it is plausible that ccf-1 signals through ELT-2 that cooperates with SKN-1 in the intestine to promote the acrylamide-induced transcriptional response. The overexpression of ELT-2 did not bypass cadmium's requirement of ccf-1 for activation of all heavy metal response genes tested, this would suggest that additional factors besides ELT-2 act downstream of ccf-1 in regulating the transcriptional response. A possibility is that some of the ccf-1 dependent cadmium genes tested may be expressed in tissues other than the intestine, and may require co-activators such as ELT-3 to facilitate stress-responsive gene expression. We hope to address this in our future work to further explore how a common requirement for ccf-1 in multiple stress responses may signal through stress-specific downstream mechanisms.

| Role of ccf-1 in longevity
Genes that regulate stress response in C. elegans are often also factors that influence aging. Our results show that the knockdown of ccf-1 shortens normal lifespan and completely suppressed the longevity of the skn-1 gain of function mutant. This is consistent with our transcriptome analysis revealing that ccf-1 dependent stress genes highly cluster to skn-1 regulated glutathione metabolism.
Interestingly in the daf-2 mutant where daf-16 is activated, the degree of lifespan extension compared to wildtype was similar when worms were fed with EV or ccf-1 RNAi. This could be interpreted as the mechanism through which ccf-1 knockdown shortens lifespan is independent of the downstream pathways through which the daf-2 mutant extends longevity. While this interpretation would conflict with evidence that skn-1 functions downstream of daf-2 mutant longevity, a possible explanation is that the knockdown of ccf-1 may not restrict other regulatory functions of skn-1 such as lipid metabolism or collagen gene expression that have been directly implicated in longevity regulation (Ewald et al., 2015;Steinbaugh et al., 2015). In support of a diminished role for skn-1 dependent xenobiotic genes in daf-2 mutant longevity, we previously showed that loss of function mutation to the xrep-4 (xenobiotics response pathways) gene that specifically attenuates skn-1 dependent gst gene expression does not reduce wildtype or daf-2(e1370) lifespan (Wu et al., 2017).
It is also possible that given the pleiotropic function of the CCR4-NOT complex in mRNA regulation, its role in lifespan regulation may be linked to its deadenylase activity not explored in this study.
Nonetheless, these data demonstrate a role for the CCR4-NOT complex as a determinant of longevity and would be a subject of interest in future studies.
Overall, our study provides evidence that the CCR4-NOT complex is a key regulator for the transcriptional response to various environmental stressors in C. elegans and highlights its role in organismal longevity and stress resistance. This finding further strengthens our collective knowledge of the eukaryotic CCR4-NOT complex with demonstrated roles ranging from transcription initiation and elongation to mRNA decay.

| Genome-wide RNAi screen and RNAi experiments
RNAi screen was performed as previously described in detail (Wu et al., 2019). Briefly, synchronized L1 QV151 larvae were obtained using hypochlorite treatment and grown in liquid nematode growth media (NGM) and fed with dsRNA producing HT115(DE3) bacteria for 2 days, followed by exposure to 100 μM cadmium chloride for 24 h and screened for suppression of numr-1p::GFP fluorescence.
Approximately 19,000 dsRNA clones from the MRC genomic RNAi feeding library (Geneservice) and the ORFeome RNAi feeding library (Open Biosystems) were screened. Clones that suppressed numr- For the pal-1 RNAi experiments, the enhanced RNAi-sensitive strain rrf-3 (pk1426) was used.

| Microscopy and fluorescent analysis
To analyze numr-1p::GFP and gpdh-1p::GFP transcriptional reporters or the CCF-1::GFP translation reporter, synchronized worms were grown on EV or ccf-1 RNAi-seeded NGM agar plates until day 1 of adulthood followed by transfer to NGM agar plates seeded with corresponding RNAi containing 100 μM of cadmium for numr-1p::GFP, 300 mM NaCl for gpdh-1p::GFP, and 100 μM of cadmium or 5 mM of acrylamide for CCF-1::GFP analysis. After 24 hours, worms were mounted on a glass slide containing a 2% agarose pad and immobilized with 2% sodium azide before imaging with a Zeiss Axioskop 50 microscope mounted with a Retiga R3 camera. Eight worms were mounted per slide and relative fluorescence was calculated using the Measure function in ImageJ followed by background subtraction.
The background signal for each image was calculated by defining an area on the same image where the fluorescence signal was absent, with the dimension of the background area subtracted constant for all images. For the ELT-2::GFP reporter, synchronized L1 worms fed with EV or elt-2 RNAi after 48 hours were imaged using the methods described above. Images were taken with a GFP filter as well as a DAPI filter to create a merged composite image to help differentiate between the ELT-2::GFP signal and intestine autofluorescence.
Methods used to assess acrylamide-induced dopaminergic degeneration via dat-1p::GFP were described previously in detail (Murray et al., 2020). Briefly, synchronized L1 C. elegans were grown on NGM agar plates seeded with EV or ccf-1 RNAi until day 1 of adulthood followed by transfer to NGM agar plates containing 5 mM of acrylamide seeded with the corresponding RNAi bacteria and grown until 6 days old. Adult worms were separated from their progeny via picking with a sterilized metal pick onto fresh acrylamide agar plates during the reproductive window. On day 6, worms were prepared for imaging using the procedures described above.
Scoring criteria for dopaminergic integrity were as follows, wildtype indicates smooth and continuous cephalic neuron (CEP) sensilla dendrite located at the anterior portion of the worm, blebbing indicates at least 1 abnormal punctae along the CEP dendrites, and breaks indicate discontinuation within the CEP dendrite. Three trials were performed with N = 15 worms scored per condition for each trial.
All grayscale images were converted to color using ImageJ, with composite images displaying both GFP and RFP/DAPI colors created using the Merge Channel function when applicable.

| Lifespan and survival assays
All assays were performed at 20°C using methods previously described (Wu et al., 2019), except for daf-2(e1370) and rrf- 3(pk1426) which were grown at 16°C during development. For lifespan assays, synchronized L1 worms obtained through the hypochlorite treatment were grown on the appropriate RNAi-seeded NGM agar plates until adulthood. One-day-old adult worms were moved to new plates and continuously transferred via daily picking during the reproductive window to accomplish progeny separation. For survival assays, synchronized L1 worms were first grown on control NGM plates seeded with appropriate RNAi bacteria until the first day of adulthood, followed by transfer to NGM plates containing either 100 μM of cadmium chloride or 5 mM of acrylamide seeded with the corresponding RNAi bacteria. For both lifespan and survival assays, the first day of adulthood is considered as 1-day-old, and worms were scored every 1-2 days for death by gentle prodding with a flamesterilized metal pick. Worms that do not respond to gentle prodding were considered dead, and worms with protruding vulva or gonad were recorded as censors. Three independent trials were formed for all assays with the number of animals scored in each condition and experiment listed in Table S3.

| RNA extraction and qPCR
The Purelink RNA mini kit (12183020; ThermoFisher) was used to isolate total RNA with a Qsonica Q55 sonicator used for tissue lysis.
Synchronized L1 N2 or rrf-3(pk1426) worms were grown on NGM agar plates seeded with EV or corresponding RNAi until day 1 of adulthood followed by transfer to either control agar plates, agar plates containing 100 μM of cadmium chloride, or agar plates containing 5 mM of acrylamide, each seeded with the corresponding RNAi bacteria for an additional 24 h followed by RNA extraction.
For each condition, N = 4 biological RNA replicates were prepared with each replicate containing ~500 worms. For qPCR, RNA was first treated with DNaseI (EN0521; ThermoFisher) followed by cDNA synthesis with the Invitrogen Multiscribe™ reverse transcriptase (4311235; ThermoFisher) using an Applied Biosystems ProFlex thermocycler. Real-time PCR (qPCR) was performed with the PowerUp™ SYBR™ Green Master Mix (#A25741) in a QuantStudio3 system.
Relative gene expression was normalized to the housekeeping gene rpl-2 (ribosomal protein large subunit) and cdc-42 (cell division cycle related), primers used for this study are shown in Table S4.

| RNA-sequencing and data analysis
Wildype N2 worms synchronized at the L1 stage were grown on NGM agar plates seeded with EV or ccf-1 RNAi until day 1 of adulthood followed by transfer to either control agar plates, agar plates containing 100 μM of cadmium chloride, or agar plates containing 5 mM of acrylamide, each seeded with the corresponding EV or ccf-1 RNAi for 24 h followed by RNA extraction. Total RNA from three biological replicates for each experimental group was extracted using the methods described above, with the exception that ~2000 to 3000 worms were used for each replicate. RNA samples were then sent to Novogen on dry ice for cDNA library construction with oligo(dT) enrichment and sequencing. Sequence annotation and data analysis were performed by Novogene.
Correlation analysis between all sequenced samples is shown in Figure S7. Given that RNA was extracted from 1-day-old adults, a potential limitation is the mixing of RNA from fertilized embryos found across all samples that may contribute to the pool of sequenced adult worm RNA.

| Y2H library screen
The full-length ccf-1 cDNA clone was generated through PCR using the Q5® High-Fidelity DNA polymerase (M0491L; New England BioLabs) and cloned into the pDEST32 vector containing the GAL4 DNA binding domain (DBD) through Gateway cloning (11789020 and 12538120; ThermoFisher) and used as the Y2H bait construct. A Y2H prey cDNA library of C. elegans genes was cloned into the pDEST22 vector containing the GAL4 activating domain (AD) using the CloneMiner™ cDNA library construction kit (A11180; ThermoFisher). A forward Y2H library screen was performed using the ProQuest Two-Hybrid System (PQ1000101; ThermoFisher) to identify protein interactors of the C. elegans CCF-1 protein.
Yeast colonies containing bait and prey constructs that grew on Trp − /Leu − /His − + 25 mM 3-Amino-1,2,4-triazole (3AT) selection plates were extracted using the Zymoprep Yeast Plasmid Miniprep II (Zymo Research, D2004) followed by Sanger sequencing for gene identification. Three independent colonies from each bait-interacting prey construct were further tested for its interaction with CCF-1 by evaluating for (1) growth on Trp − /Leu − /Ura − selection plate, (2) negative growth on Trp − /Leu − + 0.2% 5-fluoroorotic acid selection plate, (3) growth on Trp − /Leu − /His − + 25 mM AT selection plate, and (4) positive phenotype from the X-gal assay. Yeast cells were normalized to OD 600 = 0.5 for the 10 0 concentration followed by serial dilution on the growth assay. Prey construct that passed three out of four selection tests was considered a positive CCF-1 interacting protein. Yeast cells were imaged on a Bio-Rad Gel Doc EQ system.

| Statistical analyses
Graphical data and statistical analysis were performed using Graphpad Prism software (V7.04). Student's t-test was used for the comparison of two groups with the Holm-Sidak multiple test correction applied when the test is repeated for multiple genes, oneway ANOVA with Dunnett's test was used for comparison of more than two groups, two-way ANOVA with Holm-Sidak test was used for comparison of two factors, and categorical data were analyzed using the Chi-square test. Lifespan and survival data were analyzed with the log-rank test using the OASIS2 software (https:// sbi.poste ch.ac.kr/oasis 2/histo ry/). For RNA sequencing data, false discovery rate (FDR) correction was applied to determine the statistical significance. For all statistical tests, the following designations were used to indicate the p-value, *p < 0.05, **p < 0.01, ***p < 0.001.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
All datasets supporting this manuscript are found within the article and its Supporting Information. RNA-sequencing data generated from this study (raw and annotated) are available on the NCBI GEO data repository GSE194057. Strains are available upon request.