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)

Amelia Mazzone, Simon J. Gibbons, Cheryl E. Bernard, Somaira Nowsheen, Sumit Middha, Luciana L. Almada, Tamas Ordog, Michael L. Kendrick, KMarie Reid Lombardo, K. Robert Shen, Luis J V Galietta, Martin E Fernandez-Zapico, Gianrico Farrugia

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Anoctamin-1 (Ano1) is a widely expressed protein responsible for endogenous Ca2+-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.

Original languageEnglish (US)
Pages (from-to)152-163
Number of pages12
JournalFASEB Journal
Volume29
Issue number1
DOIs
StatePublished - Jan 1 2015

Fingerprint

STAT6 Transcription Factor
Interleukin-4
Exons
Transcription Factors
Genes
RNA
RNA Sequence Analysis
Mutagenesis
Chromatin Immunoprecipitation
Physiology
RNA Interference
Genetic Promoter Regions
Chromatin
Muscle
Assays
Stomach
Binding Sites
Kidney
Muscles
Neoplasms

Keywords

  • Alternative splicing
  • Calcium-activated chloride channels
  • TMEM16A

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Genetics
  • Molecular Biology

Cite this

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). / Mazzone, Amelia; Gibbons, Simon J.; Bernard, Cheryl E.; Nowsheen, Somaira; Middha, Sumit; Almada, Luciana L.; Ordog, Tamas; Kendrick, Michael L.; Lombardo, KMarie Reid; Shen, K. Robert; Galietta, Luis J V; Fernandez-Zapico, Martin E; Farrugia, Gianrico.

In: FASEB Journal, Vol. 29, No. 1, 01.01.2015, p. 152-163.

Research output: Contribution to journalArticle

Mazzone, Amelia ; Gibbons, Simon J. ; Bernard, Cheryl E. ; Nowsheen, Somaira ; Middha, Sumit ; Almada, Luciana L. ; Ordog, Tamas ; Kendrick, Michael L. ; Lombardo, KMarie Reid ; Shen, K. Robert ; Galietta, Luis J V ; Fernandez-Zapico, Martin E ; Farrugia, Gianrico. / 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). In: FASEB Journal. 2015 ; Vol. 29, No. 1. pp. 152-163.
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AU - Ordog, Tamas

AU - Kendrick, Michael L.

AU - Lombardo, KMarie Reid

AU - Shen, K. Robert

AU - Galietta, Luis J V

AU - Fernandez-Zapico, Martin E

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AB - Anoctamin-1 (Ano1) is a widely expressed protein responsible for endogenous Ca2+-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.

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