A Mediator subunit, MDT-15, integrates regulation of fatty acid metabolism by NHR-49-dependent and -independent pathways in C. elegans

Abstract

The Caenorhabditis elegans Nuclear Hormone Receptor NHR-49 coordinates expression of fatty acid (FA) metabolic genes during periods of feeding and in response to fasting. Here we report the identification of MDT-15, a subunit of the C. elegans Mediator complex, as an NHR-49-interacting protein and transcriptional coactivator. Knockdown of mdt-15 by RNA interference (RNAi) prevented fasting-induced mRNA accumulation of NHR-49 targets in vivo, and fasting-independent expression of other NHR-49 target genes, including two FA-Delta9-desaturases (fat-5, fat-7). Interestingly, mdt-15 RNAi affected additional FA-metabolism genes (including the third FA-Delta9-desaturase, fat-6) that are regulated independently of NHR-49, suggesting that distinct unidentified regulatory factors also recruit MDT-15 to selectively modulate metabolic gene expression. The deregulation of FA-Delta9-desaturases by knockdown of mdt-15 correlated with dramatically decreased levels of unsaturated FAs and multiple deleterious phenotypes (short life span, sterility, uncoordinated locomotion, and morphological defects). Importantly, dietary addition of specific polyunsaturated FAs partially suppressed these pleiotropic phenotypes. Thus, failure to properly govern FA-Delta9-desaturation contributed to decreased nematode viability. Our findings imply that a single subunit of the Mediator complex, MDT-15, integrates the activities of several distinct regulatory factors to coordinate metabolic and hormonal regulation of FA metabolism.

Figure 1.

MDT-15 specifically interacts with the NHR-49-LBD and confers activation capability in yeast. (A) Estimates of the relative interaction strength of C. elegans NHR-LBDs with MDT-15 using the yeast two-hybrid system and β-galactosidase reporter assays. Values indicate average interaction strength from quantification of six independent transformants for each plasmid combination; error bars represent SEM. The top panel shows protein expression of GAL4-DBD-NHR-LBD fusions as determined by Western blot. (B) Estimates of the relative interaction strength of two MDT-15 proteins with two independent NHR-49 clones; the schematic on top depicts MDT-15 fusion proteins. Values indicate average interaction strength from quantification of eight independent transformants for each plasmid combination; error bars represent SEM. (C) Estimate of the activation capability of the GAL4-DBD-MDT-15 fusion protein using β-galactosidase activity assay (average of six independent transformants for each plasmid); error bars represent SEM.

Figure 2.

Analysis of mdt-15 expression. (A) Relative levels of mdt-15 mRNA (normalized to ama-1 mRNA levels) in embryonic, larval, and adult N2 worms were determined by qRT-PCR. The bars represent averages of three independent experiments; error bars represent SEM. (B,C) Fluorescence and DIC micrographs of BC11928 worms at the early adult stage. The promoter of mdt-15 drives expression in the intestine (B), and in head neurons (C). Bars: B, 129 μm; C, 49 μm. (Pha) Pharynx; (Neu) head neurons; (Vul) vulva; (Int) intestine.

Figure 3.

MDT-15 regulates the expression of NHR-49-dependent and -independent FA-metabolism genes. QRT-PCR quantification of relative mRNA levels of FA-metabolism genes in fed (filled bars) or fasted (hatched bars) worms grown on control RNAi (blue), nhr-49 RNAi (red), or mdt-15 RNAi (yellow). Each bar represents the average relative mRNA level from three independent RNA isolations (normalized to ama-1 mRNA levels) from N2 L4 stage animals (except for E); error bars represent SEM. (A) Genes whose fasting-induced up-regulation depends on both NHR-49 and MDT-15. (B) Genes whose expression depends on NHR-49 and MDT-15 regardless of nutritional state. (C) Control genes. (D) MDT-15-dependent genes that are NHR-49 independent. (E) Adult-only RNAi treatment of CF512 worms.

Figure 4.

MDT-15 regulates fat storage. Fluorescence micrographs (200× magnification) reveal altered fat storage in mdt-15(RNAi) versus control(RNAi) animals, as detected by staining with the vital dye Nile Red. Dietary FAs (100 μM C20:5 and 100 μM C20:3n6) partially suppress the defect.

Figure 5.

MDT-15 governs PUFA levels and maintains the C18:0/C18:1n9 ratio. (A) The FA-desaturation/elongation pathway of C. elegans (Watts and Browse 2002). Genes highlighted in yellow are down-regulated in mdt-15(RNAi) worms (Fig. 3). (B) C18:0/C18:1n9 ratio, calculated from the data shown in C; values are indicated at the top. (C) Relative abundance of FAs, expressed as a fraction of total FA, was determined by GC/MS from N2 L4 worms grown on either control RNAi (blue), nhr-49 RNAi (red), or mdt-15 RNAi (yellow). Bars represent the average relative FA abundance from three independent experiments; error bars represent SEM.

Figure 6.

MDT-15 is necessary for normal life span and animal health. (A) RNAi treatment of CF512 worms was initiated at distinct stages to compare the effect of nhr-49 RNAi (red) and mdt-15 RNAi (yellow) on whole-life (WL) life span with the effect of RNAi only during adulthood (adult-only, AO). Mean life spans (measured in days after reaching adulthood) were 16.6 ± 0.2 d for control(RNAi) worms (blue; n = 64), 12.8 ± 0.2 d for nhr-49 AO RNAi (red; n = 42), 11.8 ± 0.1 d for nhr-49 WL RNAi (red dotted; n = 50), 11.0 ± 0.1 d for mdt-15 AO RNAi (yellow; n = 86), and 7.9 ± 0.1 d for mdt-15 WL RNAi (yellow dotted; n = 76). (B) DIC micrographs (200× magnification) of N2 worms fed control RNAi or mdt-15 RNAi; mdt-15(RNAi) worms contained vacuoles (arrows) in the intestine and vulva (Vul) area. (Right panel) Dietary FAs (100 μM C20:5 and 100 μM C20:3n6) partially suppress these defects. (C) The WL life span of CF512 worms on control RNAi (blue) and mdt-15 RNAi (yellow) was determined on plates containing H₂O or a combination of PUFAs (100 μM C20:5 and 100 μM C20:3n6), respectively. Mean life spans were 20.9 ± 0.5 d for control(RNAi) worms (blue; n = 43), 21.9 ± 0.3 d for control(RNAi) on PUFAs (blue dotted; n = 40), 9.0 ± 0.1 d for mdt-15(RNAi) worms (yellow; n = 59), and 10.5 ± 0.1 d for mdt-15(RNAi) worms on PUFAs (yellow dotted; n = 71).