Interactions between a transcription factor and its target genes clearly require the transcription factor to be expressed in the right place at the right time and the presence of the corresponding recognition elements in the target gene. Just as importantly, we need to know the molecular interactions that direct the actions of these activators and repressors to specific promoters. We are particularly interested in examining the role of REST/NRSF during neuronal differentiation. We are using a variety of molecular biological and biochemical procedures to analyse DNA / protein and protein / protein interactions that are operative during neuronal differentiation, both in vivo and in cultures of neural stem cells. This laboratory is also interested to elucidate any contribution of this factor in regulating apoptotic signaling cascades in neuronal and nonneuronal cells. The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. IGF-I up-regulated OPRM1 transcription when REST was down-regulated. We propose that a reduction in REST is a critical switch enabling IGF-I to up-regulate mu-opioid receptor expression. Another research project we are exploring regulation of transcription by bile acids in HepG2 cells. A recombinant cell line (human hepatocarcinoma HepG2) is used for assessing bile acids ability of activating the Farnesoid receptor (FXR) is co-transfected with two plasmids. The first bears the FXR. sequence to cause the cells to over-express the receptor; the second vector plasmid contains a portion of the I-BABP promoter and the firefly luciferase sequence as a reporter gene (I-BABP-Luc), so that the activity of FXR agonists or antagonists can be assessed by the luciferase expression. This system is utilized for the screening of natural and synthetic bile acids and could contribute to bile acid structure-activity relationship studies and facilitate a rational approach to the development of new drugs. It should be predictive of human pathophysiological conditions, being based on human cells, and it is rapid to carry out in comparison to animal-based tests, thus allowing the screening of many compounds.