Our Unit is interested in the molecular mechanisms underlying regulated patterns of gene expression, both in time and space. During FY 99 we have identified and characterized a functional E-BOX cis-acting element in the promoter region of the rat Arylalkylamine N-acetyltransferase (NAT) promoter that can mediate transactivation by the circadian complex CLOCK/bMAL apparently only in the retina (a master oscillator) but not in the pineal (slave oscillator). We are also continuing our efforts to identify the critical elements that constitute a circadian E-BOX by comparing the cyclin, NAT and vasopressin (AVP) E-BOXES which support different levels of transactivation by the circadian transcription complex CLOCK/bMAL (0, 8 and 40- fold increase respectively).In addition, we have successfully used the NAT promoter to drive a dominant negative form of the Fra-2 transcription factor to the rat pineal gland through a collaborative transgenesis program. This achievement has enabled us to identify the first Fra-2 target genes in vivo: Fra-2, type II deiodinase, Nectadrin (or CD24) and Id-1 (a basic- HLH class transcription factor). A second project is derived from our discovery during FY 99 of a novel gene, by a yeast two hybrid system, that interacts specifically with Period (Per) 1, one of the core components of the cellular machinery that generates the self-sustained 24 hr cycle in transcriptional output. We are currently characterizing this novel gene and its interaction with Per 1, as well as its tissue distribution and function(s). A knockout approach is also underway.
