The genetic analysis of bHLH-PAS genes in Caenorhabditis elegans
The family of basic-helix-loop-helix-PAS (bHLH-PAS) transcription factors mediate diverse cellular processes. These include several important cell fate decisions and cellular responses to environmental signals. Molecular and biochemical data indicate that C. elegans ahr-1 and aha-1 encode the orthologs of the mammalian bHLH-PAS protein, aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT). The C. elegans genome also encodes 3 other bHLH-PAS proteins, hif-1, cky-1 and T01D3.2. In this dissertation, I describe molecular, biochemical, and genetic analyses of hif-1 and aha-1 and their gene products. C. elegans hif-1 encodes the homolog of the mammalian hypoxia-inducible factor alpha subunit. They form a complex that is similar to the mammalian hypoxia-inducible factor, and hif-1 mediates hypoxic responses in C. elegans. hif-1 mutants are viable at standard laboratory conditions, but they are unable to adapt to hypoxia. Wild-type animals can survive and reproduce in 1% oxygen, but the majority of the hif-1-defective animals die in these conditions. We show that HIF-1:GFP expression is induced by hypoxia and is rapidly degraded upon reoxygenation. HIF-1 co-immunoprecipitates with AHA-1 in vitro. We conclude that the hypoxia-inducible factor complex is conserved in C. elegans. To better understand the role of aha-1 in ahr-1 and hif-1 function, I initiated a genetic analysis of aha-1 function. aha-1 deletion mutants arrest as larvae and are defective in feeding. The larval arrest phenotype of aha-1 mutants is much more severe than the ahr-1, hif-1 double mutants (which are viable), suggesting that aha-1 has additional functions. We analyzed the expression patterns of the two remaining bHLH-PAS genes, T01D3.2 and cky-1. A T01D3.2:GFP reporter is expressed in two interneurons. cky-1 may represent a new bHLH-PAS gene subfamily, and a CKY-1:GFP reporter is expressed mainly in the pharynx, the feeding organ of the worm. These data suggested that AHA-1 and CKY-1 might interact and function in the pharynx. Several lines of evidence support this model. AHA-1 and CKY-1 form a DNA-binding complex in vitro. AHA-1 is nuclear localized in the subset of pharyngeal cells that express CKY-1:GFP. Most convincingly, expression of aha-1 from the cky-1 promoter is sufficient to rescue the aha-1 larval arrest phenotype. We propose that aha-1 has an essential function in the C. elegans pharynx.