The C. elegans Myc-family of transcription factors coordinate a dynamic adaptive response to dietary restriction

Dietary restriction (DR), the process of decreasing overall food consumption over an extended period of time, has been shown to increase longevity across evolutionarily diverse species and delay the onset of age-associated diseases in humans. In Caenorhabditis elegans, the Myc-family transcription factors (TFs) MXL-2 (Mlx) and MML-1 (MondoA/ChREBP), which function as obligate heterodimers, and the forkhead box transcription factor A ortholog (PHA-4) are both necessary for the full physiological benefits of DR. However, the adaptive transcriptional response to DR and the role of MML-1::MXL-2 and PHA-4 remains elusive. We identified the transcriptional signature of C. elegans DR, using the eat-2 genetic model, and demonstrate broad changes in metabolic gene expression in eat-2 DR animals, which requires both mxl-2 and pha-4. While the requirement for these factors in DR gene expression overlaps, we found many of the DR genes exhibit an opposing change in relative gene expression in eat-2;mxl-2 animals compared to wild-type, which was not observed in eat-2 animals with pha-4 loss. We further show functional deficiencies of the mxl-2 loss in DR outside of lifespan, as eat-2;mxl-2 animals exhibit substantially smaller brood sizes and lay a proportion of dead eggs, indicating that MML-1::MXL-2 has a role in maintaining the balance between resource allocation to the soma and to reproduction under conditions of chronic food scarcity. While eat-2 animals do not show a significantly different metabolic rate compared to wild-type, we also find that loss of mxl-2 in DR does not affect the rate of oxygen consumption in young animals. The gene expression signature of eat-2 mutant animals is consistent with optimization of energy utilization and resource allocation, rather than induction of canonical gene expression changes associated with acute metabolic stress -such as induction of autophagy after TORC1 inhibition. Consistently, eat-2 animals are not substantially resistant to stress, providing further support to the idea that chronic DR may benefit healthspan and lifespan through efficient use of limited resources rather than broad upregulation of stress responses, and also indicates that MML-1::MXL-2 and PHA-4 may have different roles in promotion of benefits in response to different pro-longevity stimuli. C. elegans hermaphrodite N2 (WT), eat-2(ad465), mxl-2(tm1516), or eat-2(ad465);mxl-2(tm1516) animals were grown on dsRNA-expressing HT115 E coli for feeding-based RNAi for daf-16, pha-4, or empty-vector (EV) control. All combinations of these conditions were tested, and at least two biological replicate experiments were performed, each consisting of a separate synchronized population of animals. Animals were raised at 16 degrees C from L1 to L4, then transferred to 20 degrees C for the remainder of the experiment. Approximately 3000 total animals were collected at day 2 of adulthood for RNA isolation from populations that were synchronized at L1 and then treated with FUdR at L4 to prevent formation of progeny. Libraries for RNA-Seq were prepared and sequenced on a HiSeq2500 v4 in 1x100bp mode at the University of Rochester Genomics Research Center.