In this study we demonstrated that yeast genetic and chemical genome-wide approaches, when combined with rigorous biological follow-up, can effectively characterize a novel gene that, despite being subject to numerous large-scale phenotypic studies, had little functional annotation. Our previous work demonstrated that Crg1, a putative SAM-dependent methyltransferase, was a novel mediator of resistance to the protein phosphatase inhibitor, cantharidin 
. Here we show that the mechanism of Crg1-cantharidin interaction is through direct methylation of the compound, and that, furthermore, Crg1 plays an essential role in the cellular response to cantharidin-induced lipid alterations.
Our initial observation that cantharidin cytotoxicity is suppressed by overexpression of CRG1
suggested a specific, although not necessarily direct, cantharidin-Crg1 interaction in vivo 
. Here, we demonstrate that Crg1 is able to interact with cantharidin in vitro
, resulting in the formation of a methylated cantharidin species. Modification by methylation is known to remove negative charges on diverse molecules which can alter hydrophobicity and modulate cellular pathways and processes. Given the clear phenotype of Crg1-deficient cells and the results from our in vitro
biochemical characterization of Crg1, we hypothesize that methylation of cantharidin alters its physical properties such that it is no longer harmful to cells. In a manner similar to other methyltransferases that are known to detoxify small molecules 
, chemical modification of cantharidin provides some insight regarding how its methylation may modify its activity. For example, endothall, an unmethylated, ring-opened form of cantharidin, has been assayed for protein phosphatase inhibition 
and the methyl, ethyl, and propyl esters of endothall are still potent inhibitors of PP1 and PP2A. Several lactol derivatives of norcantharidin (the anhydride form of endothall) formed by reducing one of the carbonyl groups to a hydroxyl group have been synthesized and characterized. Modification of the free hydroxyl to form methyl, ethyl, and propyl ethers sharply reduced the ability of the drug derivatives to inhibit protein phosphatases. While the unmodified lactol form inhibited PP2A with an IC50
of 5 µM, the IC50
for the methyl ether lactol form was >1000 µM. Collectively, these observations suggest that methylation of closed-ring forms, not open-ring forms, reduces cellular toxicity. Cantharidin is more sterically hindered than norcantharidin, and as such, we would expect that its equilibrium would favor the closed-ring anhydride form more than that of norcantharidin. Accordingly, we are intrigued by the possibility that the methyl cantharidin product of the reaction catalyzed by Crg1 resembles the closed-ring lactol ether compounds that are less potent inhibitors of both growth and protein phosphatase activity. Further study to elucidate the structure of this product will enhance our understanding of how the methylation of cantharidin by Crg1 facilitates its detoxification.
Looking ahead, it will be interesting to explore if similar mechanisms of cellular detoxification in mammalian cells are mediated by methyltransferases, such as METTL7A or METTL7B, which both share sequence homology to CRG1
. Interestingly, METTL7B, also known as ALDI
, was reported to be highly expressed in kidney and liver, and associated with hepatic lipid droplets 
, providing a provocative link between Crg1-like methyltransferases cantharidin toxicity and lipid process.
In addition to characterizing the direct interaction of Crg1 with cantharidin, we investigated cellular pathways of Crg1-mediated cantharidin resistance using cells sensitized with a crg1
deletion allele. This analysis revealed that genes involved in lipid-related processes are required for survival under cantharidin-induced stress in the absence of CRG1
). Because CRG1
is both not essential and also shows very few genetic interactions under standard laboratory conditions, the identification of these genes required condition-specific assays. Our chemogenomic data are supported by lipidome-wide analysis, which demonstrated that cantharidin-induced alterations in glycerophospholipids and sphingolipids occur in a CRG1
gene dose-dependent manner. Specifically, we observed the accumulation of short chain phospholipids in the crg1Δ/Δ
mutant, suggesting that the drug affects fatty acid elongation in a Crg1-dependent fashion. Consistent with this result, we also observed that overexpression of CRG1
confers resistance to lipid stressing agents such as lithium ions and the ergosterol inhibitor fenpropimorph (Figure S9C
). Resistance to fenpropimorph is acquired by mutations in the fatty acid elongase FEN1 (ELO2)
, which is known to be involved in sphingolipid biosynthesis 
. Thus, it will be informative to test if CRG1
have overlapping functions in lipid biosynthesis.
Another possible explanation for our observation that cantharidin-induced lipidome alterations can be suppressed by increasing the gene dose of CRG1
can be found in the transcriptional changes that occur in these strains. Genes involved in methionine biosynthesis are differentially expressed in CRG1
-overexpressing strains in the presence of the drug compared to wild type and the crg1Δ/Δ
mutant. This is of particular interest because changes in methionine metabolism can regulate methylation reactions by altering levels of SAM, a methyl donor 
. For example, Tehlivets et al.
showed that defects in methionine cycling enzymes result in an imbalance of phospholipid and triacylglycerol synthesis 
. The mechanisms underlying these relationships are not yet clear, but it is possible that cells sense that the level of SAM is depleted via Crg1 activity, which results in transcriptional changes in methionine biosynthesis genes, in particular, the cystathionine beta-lyase Str3. These findings suggest that overexpression of Crg1 may buffer cantharidin-treated lipidome alterations in part through changes in the methionine cycle.
To define the ‘core’ buffering network to CRG1
in the presence of cantharidin, we compared the transcriptome and cantharidin SGA profiles. Although we did not find any obvious overlap in GO term biological processes between these datasets, in our cantharidin SGA one of the most sensitive mutants was MET22
(), a gene with a role in sulfur assimilation and methionine biosynthesis. This gene was also differentially expressed in CRG1
-overexpressing mutant vs.
wild-type strain (P
-value <0.02; Table S2
Our chemical genomics results were corroborated by traditional SGA analysis. This analysis demonstrated that CRG1
has an alleviating (suppressing) genetic interaction with RVS167
. It is well established that a similar phenotype is observed when RVS167
is deleted in combination with genes involved sphingolipid biosynthesis (e.g. SUR1
), implicating sphingolipid biosynthesis in the regulation of the actin cytoskeleton 
. Similarly to S. cerevisiae
and C. albicans
, studies in the ciliate Tetrahymena
showed that cantharidin treatment also influences PI metabolism and the actin cytoskeleton 
, demonstrating the conservation of cantharidin-lipid-actin interactions.
Understanding the transcriptional regulation of CRG1
during cantharidin stress adds many layers to the picture of the complex physiological roles of this methyltransferase. CRG1
transcription is activated by cantharidin via the conserved MAPK family components of the CWI signaling pathway 
. Hoon et al.
previously demonstrated that deletion of slt2
results in cantharidin sensitivity, suggesting that this pathway is critical for cantharidin resistance 
. In mammalian cells, several studies have reported that the MAP kinases ERK and JNK are also activated by cantharidin 
, likely as a consequence of the inhibition of protein phosphatases. Moreover, other studies reported that an intact CWI cascade is essential for maintaining lipid homeostasis 
. It remains to be determined what specific steps are involved in the activation of CRG1
by cantharidin. One possible scenario is that the CWI pathway is activated by the accumulation of aberrant lipid species in a manner analogous to previous reports that suggest that long chain bases induce the Pkc1-MAPK CWI pathway in yeast 
Based on our observations, we propose the following mechanism for Crg1-cantharidin interaction (). Cantharidin treatment inhibits PP2A and PP1, resulting in the perturbation of both lipid homeostasis and actin cytoskeleton organization. This perturbation activates the CWI pathway, which in turn induces of CRG1 transcription. The resulting Crg1 protein directly methylates cantharidin, alleviating its cytotoxicity and restoring lipid homeostasis, actin cytoskeletal architecture, as well as other cantharidin-associated effects.
In summary, our study demonstrates the value of combining classic biology approaches and chemical genomics with other “omic”-based methods for de-orphaning proteins and elucidating previously unknown mechanisms of therapeutics action.