Choe, K. P. | Search bioRxiv

bioRxiv2024

Chemical genetics in C. elegans identifies anticancer mycotoxins chaetocin and chetomin as potent inducers of a nuclear metal homeostasis response

C. elegans numr-1/2 (nuclear-localized metal-responsive) is an identical gene pair encoding a nuclear protein previously shown to be activated by cadmium and disruption of the integrator RNA metabolism complex. We took a chemical genetic approach to further characterize regulation of this novel metal response by screening 41,716 compounds and extracts for numr-1p::GFP activation. The most potent activator was chaetocin, a fungal 3,6-epidithiodiketopiperazine (ETP) with promising anticancer activity. Chaetocin activates numr-1/2 strongly in the alimentary canal but is distinct from metal exposure because it represses canonical cadmium-responsive metallothionine genes. Chaetocin has diverse targets in cancer cells including thioredoxin reductase, histone lysine methyltransferase, and acetyltransferase p300/CBP; further work is needed to identify the mechanism in C. elegans as genetic disruption and RNAi screening of homologs did not induce numr-1/2 in the alimentary canal and chaetocin did not affect markers of integrator dysfunction. We demonstrate that disulfides in chaetocin and chetomin, a dimeric ETP analog, are required to induce numr-1/2. ETP monomer gliotoxin, despite possessing a disulfide linkage, had almost no effect on numr-1/2, suggesting a dimer requirement. Chetomin inhibits C. elegans growth at low micromolar levels and loss of numr-1/2 increases sensitivity; C. elegans and Chaetomiaceae fungi inhabit similar environments raising the possibility that numr-1/2 functions as a defense mechanism. There is no direct ortholog of numr-1/2 in humans, but RNAseq suggests that chaetocin affects expression of cellular processes linked to stress response and metal homeostasis in colorectal cancer cells. Our results reveal interactions between metal response gene regulation and ETPs and identify a potential mechanism of resistance to this versatile class of preclinical compounds.

pharmacology and toxicology↗

bioRxiv2022

CCR4-NOT subunit CCF-1/CNOT7 interacts with the PAL-1/CDX-1 transcription factor to regulate multiple stress responses in Caenorhabditis elegans

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 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 glutathione s-transferases. Consistently, survival assays show that knockdown of ccf-1 decreases C. elegans stress resistance. 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 pal-1 down-regulates the mRNA levels of elt-2 and elt-3, which serves as the master transcriptional co-regulators of stress response in the C. elegans intestinal and epidermal tissues respectively. These results reveal a new role for CCR4-NOT in stress response regulation with PAL-1 through the transcriptional control of elt-2 and elt-3 in C. elegans.

genetics↗