title: Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6).
abstract: Anoctamin-1 (Ano1) is a widely expressed protein responsible for endogenous Ca(2+)-activated Cl(-) currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next-generation RNA sequencing (RNA-seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1-fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL-4, increased promoter activity by 1.6 ± 0.02-fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL-4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12-fold increase in binding of STAT6 to P0 after IL-4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases.
authors.affiliation: Enteric NeuroScience Program.
authors.forename: Amelia
authors.initials: A
authors.lastname: Mazzone
authors.affiliation: Enteric NeuroScience Program.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gibbons
authors.affiliation: Enteric NeuroScience Program.
authors.forename: Cheryl E
authors.initials: CE
authors.lastname: Bernard
authors.affiliation: Mayo Medical School.
authors.forename: Somaira
authors.initials: S
authors.lastname: Nowsheen
authors.affiliation: Biomedical Statistics and Informatics.
authors.forename: Sumit
authors.initials: S
authors.lastname: Middha
authors.affiliation: Schulze Center for Novel Therapeutics, Department of Oncology, and.
authors.forename: Luciana L
authors.initials: LL
authors.lastname: Almada
authors.affiliation: Enteric NeuroScience Program.
authors.forename: Tamas
authors.initials: T
authors.lastname: Ordog
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA; and.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Kendrick
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA; and.
authors.forename: K Marie
authors.initials: KM
authors.lastname: Reid Lombardo
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA; and.
authors.forename: K Robert
authors.initials: KR
authors.lastname: Shen
authors.affiliation: U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, Italy.
authors.forename: Luis J V
authors.initials: LJ
authors.lastname: Galietta
authors.affiliation: Schulze Center for Novel Therapeutics, Department of Oncology, and.
authors.forename: Martin E
authors.initials: ME
authors.lastname: Fernandez-Zapico
authors.affiliation: Enteric NeuroScience Program, farrugia.gianrico@mayo.edu.
authors.forename: Gianrico
authors.initials: G
authors.lastname: Farrugia
mesh_terms: D000075369:Anoctamin-1; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018118:Chloride Channels / Q000235:genetics*; D019175:DNA Methylation; D005091:Exons; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D015847:Interleukin-4 / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D050800:STAT6 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D018118:Chloride Channels; C508598:IL4 protein, human; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015847:Interleukin-4
keywords: TMEM16A; alternative splicing; calcium-activated chloride channels