Identification of DAF-16/FoxO targets associated with longevity. Identification of DAF-16/FoxO targets associated with longevity

FoxO transcription factors can promote longevity in invertebrates and mammals. In C.elegans, the FoxO family member DAF-16 is required for lifespan extension in the contexts of daf-2/IGFR mutation and germline ablation. The daf-16 genomic locus encodes three distinct groups of transcripts (a,b, and d/f/h). In animals with reduced insulin signaling or ablated germlines, mutations that reduce daf-16a and d/f/h levels without affecting daf-16b reduce lifespan to the same extent as daf-16 null mutations. We reasoned that identifying a set of targets of the daf-16a and d/f/h isoforms that are differentially regulated in two contexts of reduced insulin signaling and in germline ablated animals would enrich for the daf-16 transcriptional targets that are required for longevity. We identified targets that were differentially regulated in daf-2(e1368) and daf-2(e1370) mutants relative to wild-type, and differentially regulated in the opposite direction from WT in compound mutants with both daf-16(mg54), which eliminates the a and d/f/h isoforms, and daf-16(mu86) which is a predicted null allele. We performed a similar analysis in glp-1(e2141) germline ablated animals (omitting the wild-type comparison). We then identified daf-16 targets associated with longevity (dal genes) which were found in all three sets of comparisons. Overall design: 10 strains were profiled, with 5 biological samples of each strain, for a total of 50 samples. Synchronized populations were grown to day 1 of adulthood, then harvested for RNA. Animals were grown at 15C for the first 72 hours to permit dauer bypass, then upshifted to 20C, except for glp-1 strains which were grown at 25C to ablate the germline. Lifespan analysis was performed on a cohort from each sample to confirm expected phenotypes. Strains were wild-type (WT), daf-2(e1370), daf-16(mg54);daf-2(e1370), daf-16(mu86);daf-2(e1370), daf-2(e1368), daf-16(mg54);daf-2(e1368), daf-16(mu86);daf-2(e1368), glp-1(e2141), daf-16(mg54);glp-1(e2141), daf-16(mu86);glp-1(e2141).

FoxO转录因子（FoxO transcription factors）可促进无脊椎动物与哺乳动物的寿命延长。在秀丽隐杆线虫（C.elegans）中，FoxO家族成员DAF-16是daf-2/胰岛素样生长因子受体（IGFR）突变以及生殖细胞消融条件下寿命延长所必需的蛋白。daf-16基因组位点编码三类不同的转录本（a、b及d/f/h亚型）。在胰岛素信号减弱或生殖细胞被消融的线虫个体中，仅降低daf-16a与d/f/h的表达水平而不影响daf-16b的突变，其对寿命的缩短效果与daf-16完全功能缺失（null）突变相当。我们推测，鉴定在两种胰岛素信号减弱情境以及生殖细胞消融动物中差异调控的daf-16a和d/f/h亚型的靶标集合，将富集到寿命延长所必需的daf-16转录靶标。我们鉴定出相对于野生型（WT），在daf-2(e1368)与daf-2(e1370)突变体中差异表达的靶基因，且这些靶基因在同时携带可消除a与d/f/h亚型的daf-16(mg54)与预测为完全功能缺失等位基因的daf-16(mu86)的复合突变体中，呈现与野生型相反的调控方向。我们在glp-1(e2141)生殖细胞消融线虫中开展了类似分析（省略野生型对照比较步骤）。最终我们鉴定到与寿命延长相关的daf-16靶标（daf-16 targets associated with longevity，简称dal基因），这类靶标在全部三组比较分析中均被检测到。实验整体设计：共对10个菌株开展转录组谱分析，每个菌株设置5个生物学重复样本，总计50个样本。将同步化的线虫种群培养至成虫第1天，随后收集样本提取RNA。所有线虫在15℃下培养前72小时以绕过持久态（dauer），之后除glp-1菌株外均转移至20℃培养；glp-1菌株则在25℃下培养以实现生殖细胞消融。从每个样本对应的线虫队列中开展寿命分析，以验证预期的表型。所用菌株包括：野生型（WT）、daf-2(e1370)、daf-16(mg54);daf-2(e1370)、daf-16(mu86);daf-2(e1370)、daf-2(e1368)、daf-16(mg54);daf-2(e1368)、daf-16(mu86);daf-2(e1368)、glp-1(e2141)、daf-16(mg54);glp-1(e2141)以及daf-16(mu86);glp-1(e2141)。