To identify genetic contributions to alcoholism vulnerability, we focused on serotonergic behaviors, since a subtype of alcoholism is associated with decreased serotonin turnover. Serotonin biosynthesis is governed by tryptophan hydroxylase (TPH), which is rate-limiting. We hypothesize that factors controlling its gene expression play a major role in behavior. The promoter elements controlling TPH gene expression have been analyzed through the use of gene fusions and EMSAs. Two regions necessary for high level transcription were identified; two sites in the upstream TPH promoter and a site in the 5' untranslated region bind Sp1. An upstream site was found to be required for repression in a cell- specific fashion and bound RBP-Jk. TPH is the first mouse gene shown to be regulated by RBP-Jk. RBP-Jk probably repressed TPH transcription by quenching Sp1 activation through blockage of Sp1 function. A variant RBP-Jk allele, carrying an amino acid substitution, located within the DNA-binding domain was discovered. High affinity binding sites for RBP- Jk were identified among 5 introns (introns 1, 2, 6, 7 and 10) of the mouse TPH gene. Several RBP-Jk were also found in the human TPH gene. TPH gene expression has been studied in vitro to define the mechanisms controlling its regulation. We hypothesized a negative feedback-loop regulating serotonin production. TPH-luciferase constructs have been used to identify several agents which regulate TPH transcription. Furthermore, TPH mRNA was demonstrated in Neuro-2a cells, the first report in a neuronal line. Experiments are in progress in the rat with direct injections of TPH and 5-HT antisense oligothiophospho- nucleotides directly into the raphe nucleus. The role of TPH and 5-HT in alcohol consumption is being investigated.
