HEPATOCYTE NUCLEAR FACTOR-1-ALPHA REGULATES TRANSCRIPTION OF THE GUANYLIN GENE

To study the molecular mechanisms controlling guanylin expression, we have cloned the mouse guanylin gene, including 2.7 kb of upstream sequ ence. We show that the first 133 base pairs (bp) of the upstream guany lin promoter are sufficient to drive near maximal (6-fold over basal) luciferase reporter gene expression in Caco-2 intestinal cells; at lea st 300 bp of upstream promoter are required for reporter gene expressi on in HT-29 intestinal cell Lines. Using electromobility shift assays, we demonstrate that nuclear proteins bind to the hepatocyte nuclear f actor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5' flanking region, is r equired for transcriptional activation of the guanylin gene in both in testinal cell lines. Mutagenesis of the HNF-1 consensus sequence aboli shes transcriptional activation of guanylin promoter-luciferase report er gene constructs. Cotransfection of these constructs with HNF-1 alph a augments transcriptional, initiation of the reporter gene. In contra st, HNF-1 beta has no significant effect on transcription of the repor ter gene. These experiments demonstrate that HNF-1 alpha is an importa nt regulatory element in the transcriptional activation of guanylin.