The Human Pendrin Promoter Contains two N₄GAS Motifs with Different Functional Relevance

Abstract

Background: Pendrin, an anion exchanger associated with the inner ear, thyroid and kidney, plays a significant role in respiratory tissues and diseases, where its expression is increased following IL-4 and IL-13 exposure. The mechanism leading to increased pendrin expression is in part due to binding of STAT6 to a consensus sequence (N₄ GAS motif) located in the pendrin promoter. As retrospective analyses of the 5' upstream sequence of the human pendrin promoter revealed an additional N₄ GAS motif (1660 base pairs upstream of the one previously identified), we set out to define its contribution to IL-4 stimulated changes in pendrin promoter activity. Methods and Results: Electrophoretic mobility shift assays showed that STAT6 bound to oligonucleotides corresponding to both N₄ GAS motifs in vitro, while dual luciferase promoter assays revealed that only one of the N₄ GAS motifs was necessary for IL-4 -stimulated increases in pendrin promoter activity in living cells. We then examined the ability of STAT6 to bind each of the N₄ GAS motifs in vivo with a site-specific ChIP assay, the results of which showed that STAT6 interacted with only the N₄ GAS motif that was functionally implicated in increasing the activity of the pendrin promoter following IL-4 treatment. Conclusions: Of the two N₄ GAS motifs located in the human pendrin promoter region analyzed in this study (nucleotides -3906 to +7), only the one located nearest to the first coding ATG participates in IL-4 stimulated increases in promoter activity.

Interleukin-4 Induces CpG Site-Specific Demethylation of the Pendrin Promoter in Primary Human Bronchial Epithelial Cells

“…We chose to focus on six CpG sites (91, 90, 89, 87, 86 and 85) within the pendrin promoter due to their proximity to the N 4 GAS motif that is essential for the response to IL-4 [21, 23]. The basal and IL-4 stimulated methylation patterns for these CpG sites in all four cell types are shown in Fig.…”

“…This upregulation is induced by phosphorylated STAT6, a transcription factor activated by IL-4, which recognises and binds to a functional N 4 GAS motif present in the pendrin promoter [21, 23]. Several CpG sites, hotspots for DNA methylation, are located between the binding motif for STAT6 and the transcription start site of the pendrin gene.…”

“…One of the main downstream effectors activated by IL-4 and IL-13 is the signal transducer and activator of transcription, STAT6. Activation of this transcription factor by phosphorylation has been shown to be directly responsible for increased pendrin mRNA expression stimulated by IL-4 through interaction with a functional N 4 GAS motif present in the pendrin promoter [21, 23]. …”

The Significance of Hypothiocyanite Production via the Pendrin/DUOX/Peroxidase Pathway in the Pathogenesis of Asthma

“…Since the SLC26A4 gene is an IL-4/IL-13-inducible molecule, it was reasonable to think that STAT6, a transcriptional factor critical for the IL-4/IL-13 signals, regulates the expression of the SLC26A4 gene. Nofziger et al found that there exist two consensus binding sites for STAT6 (TTC(N 4 )GAA) at −3472 to −3463 (motif 1) and −1812 to −1803 (motif 2) of the 5′-flanking region of the SLC26A4 gene [ 24 , 25 ]. Vanoni et al showed that although both consensus sequences can bind STAT6 following IL-4 exposure, IL-4- or IL-13-inducible pendrin expression requires only motif 2 [ 25 ].…”

“…Nofziger et al found that there exist two consensus binding sites for STAT6 (TTC(N 4 )GAA) at −3472 to −3463 (motif 1) and −1812 to −1803 (motif 2) of the 5′-flanking region of the SLC26A4 gene [ 24 , 25 ]. Vanoni et al showed that although both consensus sequences can bind STAT6 following IL-4 exposure, IL-4- or IL-13-inducible pendrin expression requires only motif 2 [ 25 ]. These results suggest that IL-4 or IL-13 induces expression of the SLC26A4 gene in a cis -regulating manner.…”