Figure 7. ins-7 and other unc-62-dependent class N targets are activated in the old intestine.

(A) Class N unc-62 targets tend to increase with age in the intestine. Eight class N targets (bound by UNC-62 in the young adult with neuronal-enriched expression in development (dark grey bars)), as well as ten control targets that have neuronal-enriched expression and are bound by UNC-62 in L3 stage but not the adult stages (light grey bars), were assayed for age-related changes in the intestine (between day 1 and day 15 adults). Expression in dissected intestines was quantified by qPCR as described in Methods using a beta-tubulin control (C36E8.5). Bars indicate the ratio of the expression between young and old as measured by the ΔΔCt method, and error bars indicate standard deviation of technical triplicate qPCR measurements. + indicates transcripts that were quantified in young but not detected in old intestines; for these transcripts, age-downregulation was calculated using a Ct value of 40 as an upper bound of old intestinal expression. (B) unc-62 activates four of five class N targets in old intestines. For the five UNC-62 adult targets that increased with age by more than two-fold, we analyzed expression in day 1, day 15 control, and day 15 unc-62 RNAi micro-dissected intestines (two biological replicate samples each). (Left) Schematics for these five genes indicates gene structures (in blue), as well as associated UNC-62 binding sites in young adult (red) ChIP-seq. All binding sites shown were factor-specific and significant at q-value 10⁻⁵ (**). (Right) Expression fold-change was calculated using a beta-tubulin control as before, and are shown relative to the average expression between young intestine replicates. For four of the five (tsp-1, max-1, ins-7, and T05B11.1), expression in old intestines was diminished when unc-62 was knocked down. unc-62 RNAi had no effect on stdh-1 expression in old intestines. Although the binding site overlapping the 3′ end of tsp-1 was associated with tsp-1 using our analysis pipeline, we found that tsp-2 showed a similar expression pattern in dissected intestines (Figure S10). (C) unc-62 RNAi activates activity of main insulin pathway target DAF-16/FOXO. We quantified expression of sod-3:GFP, which is a reporter of DAF-16 activity [40]. Approximately 25 worms were imaged for each aging timepoint (x-axis), and fluorescence in the head (y-axis) was quantified using ImageJ. Error bars indicate standard error of the mean. (D) unc-62 RNAi extends lifespan in wild-type worms (37% increase in mean lifespan, p<10⁻⁵), but not in daf-16(m26) mutants (3% increase, p>0.1). X-axis indicates days of adulthood, and the y-axis indicates percent of worms remaining alive. Similar results were observed in a replicate experiment using daf-16 RNAi (lifespan data in Table S2).