Su2023 IL-4 via STAT6 Regulates a Promoter for the Human Ano1 Gene

Background: Ano1 is a Ca2+ activated Clchannel expressed in interstitial cells of Cajal (ICC) in the smooth muscle layers of the GI tract. Ano1 has been shown to play an important role in the electrical activity of ICC, where it is recognized as the primary channel responsible for the endogenous Ca2+-activated Clcurrent, and also plays a role in ICC proliferation. Multiple isoforms have been described for Ano1 with diverse electrophysiological properties. Relative expression levels of isoform are altered in diabetic gastroparesis thus potentially affecting the function of the channel, however the mechanisms underlying the differential expression of Ano1 are not known. Recently, we identified a region with promoter activity adjacent to a new exon of human Ano1 upstream of the published exon 1. Aim: The aim of the present study was to characterize the Ano1 promoter to gain insight into the regulatory mechanisms governing the expression of Ano1. Methods: Consensus sequences for core promoter and potential regulatory elements were identified in silico. A luciferase reporter assay was used for the evaluation of the activity of the wild-type and mutant promoters and chromatin immunoprecipitation (ChIP) assays were used to characterize the recruitment of RNA polymerase II (PolII) and transcription factors of interest to Ano1 promoter. Results: When expressed in HEK293 cells, the putative promoter region representing 1.8 kb upstream of the novel transcription start site (TSS) drove a significant increase in the luciferase activity under basal conditions (6.7±2.1 fold). IL-4, a known regulator of Ano1 expression, further increased promoter activity (1.6 ± 0.02 fold over vehicle control). Using deletion mutant constructs we identified a 405 bp region as the minimal functional promoter. Bioinformatics analyses identified core promoter elements in this region. No TATA box was found but there was an initiator element (INR) 30 bp upstream of the TSS, as well as a CpG island. ChIP assay using antibodies against the activated form of PolII, phosphorylated on serine 5, confirmed that the TSS identified by 5' RACE was functional and located 202 bp upstream of the translational start codon. The promoter region contains putative binding sites for multiple transcription factors including STAT6, a downstream effector of IL-4 signaling. A combination of luciferase experiments, site directed mutagenesis, siRNA knockdown and ChIP confirmed the involvement of STAT6 in the IL-4 regulation of the Ano1 gene. Conclusions: In conclusion, a promoter for the human Ano1 gene identified upstream of a newly described exon drives Ano1 expression and is modulated by IL4 via STAT6. Identification of the human Ano1 promoter and its regulation allows further understanding of the regulation of Ano1 and changes in expression associated with disease. Supported by NIH grant DK57061.