Direct repression of anoctamin 1 ( <i>ANO1</i> ) gene transcription by Gli proteins

Abstract

The Ca²⁺-activated Cl^- channel, anoctamin 1 (Ano1, also known as transmembrane protein 16A) contributes to intestinal pacemaking, fluid secretion, cellular excitability, and tissue development. The human <i>ANO1</i> promoter contains binding sites for the glioma-associated oncogene (Gli) proteins. We investigated regulation of <i>ANO1</i> transcription by Gli. <i>ANO1</i> promoter activity was determined using a luciferase reporter system. Binding and functional effects of Glis on <i>ANO1</i> transcription and expression were demonstrated by chromatin immunoprecipitation, small interfering RNA knockdown, PCR, immunolabeling, and recordings of Ca²⁺-activated Cl^- currents in human embryonic kidney 293 (HEK293) cells. Results from previous genome-wide association studies were used to test <i>ANO1</i> promoter polymorphisms for association with disease. Gli1 and Gli2 bound to the promoter and repressed <i>ANO1</i> transcription. Repression depended on Gli binding to a site close to the <i>ANO1</i> transcriptional start site. Mutation of this site prevented Gli binding and transcriptional repression. Knockdown of Gli expression and inhibition of Gli activity increased expression of <i>ANO1</i> RNA and Ca²⁺-activated Cl^- currents in HEK293 cells. A single-nucleotide polymorphism prevented Gli binding and showed association with irritable bowel syndrome. We conclude that Gli1 and Gli2 repress <i>ANO1</i> by a novel mechanism that is independent of Gli cleavage and that has a role in gastrointestinal function.-Mazzone, A., Gibbons, S. J., Eisenman, S. T., Strege, P. R., Zheng, T., D'Amato, M., Ordog, T., Fernandez-Zapico, M. E., Farrugia, G. Direct repression of anoctamin 1 (<i>ANO1</i>) gene transcription by Gli proteins.