While studying the role of Mediator in EGFR signaling in C. elegans, we observed striking differences in Muv penetrance of mdt-13 mutants fed different bacterial food sources: mdt-13 mutants fed HT115 transformed with an empty RNAi vector (HT115 + RNAi(EV)) showed significantly lower Muv penetrance than those fed OP50 (Fig. 1A).
All 4 Mediator kinase module subunits are required to repress vulval organogenesis in C. elegans, but mdt-12 and mdt-13 mutants display a much more penetrant Muv phenotype than cdk-8 or cic-1 mutants. Unlike the effect seen in mdt-13 mutants, the Muv penetrance of the cdk-8 mutant did not change significantly on HT115 + RNAi(EV) vs. OP50 (Fig. 1A); however, this could be due to the very low penetrance of the Muv phenotype in the cdk-8 mutant (Fig. 1A). Therefore, for further investigation, we utilized a sensitized genetic background, cdk8; lin-15A, which shows enhanced Muv penetrance compared to the cdk-8 single mutant.
Using mdt-13 and cdk-8; lin-15A double mutants, we tested whether the HT115 bacterial food source itself, rather than the RNAi(EV) vector or the selective media used for RNAi experiments, was responsible for the observed changes in mdt-13 Muv penetrance (Fig. 1A). Indeed, mdt-13 mutants fed untransformed HT115 bacteria and grown on standard media displayed a substantially and significantly reduced Muv penetrance compared to mdt-13 mutants fed OP50 (Fig. 1B). We observed a similar effect in cdk-8; lin-15A mutants (Fig. 1B). Thus, different E. coli strains can modulate ectopic vulval organogenesis in two Mediator kinase module mutants.
C. aquatica has profound effects on the developmental rate of C. elegans, and, like an HT115 diet, completely suppresses the sterility of nhr-114 mutants. Therefore, we reasoned that C. aquatica might also affect the formation of ectopic vulvae during development. Surprisingly, we observed no change in Muv penetrance of mdt-13 or cdk-8; lin-15A mutants fed C. aquatica vs. OP50 (Fig. 1B). This experiment demonstrates that 2 strains of E. coli compared in this study, OP50 and HT115, can have more disparate effects on vulva formation in Mediator kinase module mutants than species from two different bacterial genera, E. coli OP50 vs. C. aquatica.
The results presented here demonstrate that the bacterial diet of C. elegans can influence ectopic vulva formation in Mediator kinase module subunit mutants. Ectopic vulva formation in these mutants arises due to derepression of EGFR-Ras-ERK signaling-driven cell fates. Thus, our findings imply that the worm diet may modulate EGFR-Ras-ERK signaling pathway activity, at least in Mediator kinase module mutants. As human Mediator kinase module subunit mutations have been identified in several tumor types, it would be of interest to investigate whether such tumors are sensitive to changes in nutrient availability.
The nutritional signals that alter vulval organogenesis in Mediator kinase module mutants remain unidentified, but comparison of lipid composition of OP50 vs. HT115 may provide some clues. OP50 is higher in TAGs than HT115, which causes accumulation of TAGs in C. elegans fed OP50 compared to HT115. Interestingly, the TAG content of the C. elegans diet correlates inversely with the accumulation of branched-chain fatty acids (BCFAs) in worms. Feeding mice a high-fat diet has been linked to a decrease in short chain fatty acids (SCFAs), which are precursors of BCFAs. This decrease in SCFAs alters the microbiota composition of the intestinal tract of mice expressing a Ras gain-of-function allele in the intestine (RasG12Dint), and intestinal dysbiosis increases tumor incidence in RasG12Dint mice via activation of immune signaling. It is thus tempting to speculate that the differences we observed in Muv penetrance of kinase module mutant worms, akin to tumor incidence in mice, are due to alterations in BCFAs caused by a high-fat diet, which may favor activation of an intestinal immune response. Clearly, many aspects of this model must be tested, such as whether EGFR-Ras-ERK signaling-driven cell fates occur at higher incidence in worms fed OP50 vs. HT115, whether TAGs are the main driver of the observed phenomenon, and whether blocking an immune response can reverse the observed effects of OP50. If these predictions hold true, then it will provide compelling evidence to test whether human tumor cell lines with mutations in Mediator kinase module subunits are responsive to nutritional cues.