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

DANIEL HOCHBAUM; ALFRED L. FISHER

Encuentro; University of Pittsburgh Science day; 2009

The free living soil nematode C elegans develops through four larvae stages, until becomes an adult. When environmental conditions are not favorable (ie: high temperature, high population and nutrient limitations), animals will arrest at the dauer diapause, a long-lived resistant structure. When conditions are favorable again, animals will resume development. A key regulator of dauer formation is the nuclear receptor Daf-12, a C elegans homolog of the vitamin D receptor. Like a classical nuclear receptor, Daf-12 contains an N-terminal DNA binding domain and a C-terminal ligand binding domain, wich docks coactivator and corepressor complexes. Dependent on the type of mutation within Daf-12, animals can exhibit a constitutive daf phenotype (daf-c), as well as daf defective phenotype (daf-d). As a general model, in favorable environment, both Insulin and TGF-β secretion are required to produce dafrachonic acid (DA), the Daf-12 ligand. Upon DA binding, it is believed that Daf-12 recruits a coactivator, necessary to regulate genes involved in development. On the other hand, when neither Insulin nor TGF-β are present, Daf-12 binds to a corepressor called Din-1. This interaction leads to dauer arrest. Although some of the signals upstream Daf-12 is known, which genes are regulated by this receptor are largely unknown. In an attempt to find genes modulated by Daf-12, a transgenic worm expressing a Tap-tagged Daf-12 was generated. A chip on chip assay was performed, leading to the identification of several binding sites. Among several genes found, Daf12 might be regulating expression of heterochronic genes, neccesary control the expression of stage-specific developmental events. To our surprise, Daf-12 showed several binding sites within its corepressor Din-1. Indeed, Daf-12 also showed interaction with cbp-1, the C elegans homolog of the coactivator CBP, suggesting that Cbp-1 could act as a Daf-12 coactivator. To validate our chip on chip results, real time PCR were performed to quantify some of the target genes in a Daf-12 deficient strain. Also, different trans-genes carrying a specific promoter fused to GFP were introduced in WT or Daf-12 deficient worms to compare expression patterns. Last, in an attempt to reconstitute in vitro transcriptional activity, HEK cells were transfected with luciferase fusion reporters and Daf-12. Upon agonist stimulation (DA), luciferase activity was measured.