Identification of C. elegans NHR target genes via chIP-chip

DANIEL HOCHBAUM; ALFRED L. FISHER

Congreso; Aging, Metabolism, Stress, Pathogenesis, and Small RNAs; 2010

The C. elegans NHR daf-12 shows sequence homology to the vertebrate LXR and vitamin D receptors, and is involved in development, aging, and control of entry into the alternative dauer larval stage during adverse conditions. The daf-12 receptor is regulated by a class of ligands termed dafachronic acids which are a group of carboxylated steroids. The dafachronic acids are produced from cholesterol via a multi-step pathway involving the daf-36 Rieske-like oxygenase and the daf-9 cytochrome P450 enzyme. While the biologic roles and hormonal regulation of daf-12 are becoming better understood, the identities of target genes for daf-12 are still largely unknown. To identify target genes for daf-12, we developed transgenic animals which express an epitope-tagged transgene, and we then used these worms to perform chromatin immunoprecipitation followed by hybridization of the precipitated DNA to whole genome tiling arrays. From the arrays, we identified 1175 genomic regions bound by daf-12 in three independent samples, and these regions are within 5 kb of 3179 genes. Importantly, these 3179 genes include known target genes such as lit-1, myo-2, and the microRNA genes mir-84, and -241 which are regulated by daf-12. Among the genes, we identified are genes involved in stress resistance (heat shock proteins, hsf-1, skn-1), dauer formation (daf-16, daf-3), and metabolism. We also find novel roles for daf-12 in the regulation of circadian rhythms, as the circadian clock genes lin-42, aha-1, kin-20, and kin-19 all are near regions identified on the arrays, and miRISC complex expression, as the alg-1, alg-2, ain-1, cgh-1, and nhl-2 genes are also putative daf-12 target genes. To further explore the role of daf-12 in regulating circadian rhythms, we tested the effects of dauer formation of the expression of lin-42 and kin-20 and found significant repression of both via either Q-PCR or transgenic worms. Together, our results suggest that we have successfully identified novel target genes for the daf-12 NHR, and have identified a novel role for NHR´s in the regulation of microRNA function.