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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 cells showed a 6.
Treatment with a known regulator of Ano1 expression, IL-4, increased promoter activity by 1. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 STAT6a downstream effector of IL Chromatin immunoprecipitation ChIP experiments on T84 cells, which endogenously express Ano1, showed a 2.
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—Mazzone, A.
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. A no 1 TMEM16A, anoctamin1 is one of a family of genes encoding membrane proteins with 8 transmembrane spanning domains known as anoctamins. Expression of Ano1 has been described in a broad range of tissues, including a variety of epithelia 13 — 6sensory cells 378and smooth muscles 9 — In the muscle layers of the human and mouse 113001 tract, Ano1 is not present in smooth muscle cells but is expressed exclusively in the interstitial cells of Cajal Functionally, Ano1 plays a critical role in many physiologic processes, including chloride transport in airways 19salivary glands 34and gastrointestinal epithelial cells 14 ; rhythmic contraction 1516 and proliferation 1718 in the gastrointestinal tract; and heat sensation in aj neurons Thus, it is not surprising that Ano1 knockout mice do not survive long past weaning 9.
Aberrant expression and activity of Ano1 have also been implicated in the pathophysiology of several diseases, including cancer 20 — 23cystic fibrosis 24and hypertension 25 — It has been identified as a possible alm target in cystic fibrosis and asthma 2428as well as hypertension-associated 13001 diseases such as stroke 2526 and pulmonary hypertension The widespread expression and diverse functions of the Ano1 protein indicate that expression of the gene must be controlled by several mechanisms.
For example, in the lung, IL-4 is a potent inducer of Ano1 expression 1 and may be important in the pathophysiology of asthma In several cancers, Ano1 expression is also highly up-regulated by mechanisms that are not fully established 20 — An additional complication in understanding the transcription of the Ano1 gene is the variety of splice variants distributed across different tissues and in different physiologic and pathologic situations.
The published Ano1 gene comprises 26 exons coding for multiple splice variants in both human 130 and mouse These isoforms had different electrical properties, and changes to the expression of these different transcripts were found in the disease gastroparesis, characterized by delayed stomach emptying Although there is abundant evidence a,j the role of Ano1 dysregulation in the pathogenesis of different diseases, the mechanisms underlying the differential expression of this ion channel and its splicing variants in health and in disease are still elusive.
The goal of this study was to identify and characterize the human Ano1 promoter to further understand the regulation of Ano1 in diverse cell types and changes in expression associated with disease. Bioinformatics, luciferase, expression, and ChIP assays demonstrate that the promoter drives Ano1 expression and is modulated by IL-4, the best characterized activator of Ano1, via the STAT6 transcription factor. Strips of human gastric smooth muscle were obtained from 8 nondiabetic patients undergoing duodenal switch gastric bypass surgery for obesity following institutional review board-approved protocols.
Approximately 5 kb of sequence upstream of the newly identified exon 0 of human ANO1 was analyzed using 2 bioinformatics tools for the recognition of human RNA Polymerase II PolII promoter regions and start of transcription.
Neural Network Promoter Prediction www. For the full-length P0 reporter vector and all the truncation reporter plasmids, the fragments were amplified from human genomic DNA by PCR using specific primers Table 1 and cloned into the pMetLuc vector included in the Ready-To-Glow secreted luciferase reporter system Clontech using the Eco RI restriction site.
The integrity of the constructs and the presence of the desired mutation were verified by DNA sequencing. The primers used are listed in Table 2.
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Sequences of primers aln for cloning of P0 promoter and deletion constructs into luciferase reporter vector. After 4 h, the medium was changed to serum containing medium, and the cells were let rest for an additional 2 h. They were then lifted and divided into 3 wells for each condition. To assess the activity of P0 as a promoter, the Ready-To-Glow secreted luciferase reporter system Clontech was used, according to the manufacturer instructions. This system uses secreted Metridia luciferase as a reporter molecule to monitor the activity of promoters and enhancers by sampling media supernatant, without the need for cell lysis.
This vector expresses secreted embryonic alkaline phosphatase SEAPa secreted form of human placental alkaline phosphatase, as a reporter molecule. Luciferase assays were performed in HEK cells, because T84 cells had endogenous high expression of human placental 130001 phosphatase that made them unsuitable for the SEAP assay used for normalization.
The primers used were designed using the Bisulfite Primer Seeker Program www. After PCR, the bands alm gel purified, a,j using the TA cloning kit Life Technologiesand sequenced to analyze the presence of converted cytosines; 20—50 clones per sample ajl analyzed. ChIP was performed as described previously Quantitative PCR was performed for each sample as described below and experiments were repeated 3 times.
All the primers used are listed in Table 4.
Beta-actin was used as the housekeeping gene expression control Qiagen. The sequences of all the primers used for the quantitative PCR experiments are listed ali Table 4. Currents were recorded by standard whole-cell voltage clamp recordings from HEK cells transfected with either of the Ano1 isoforms examined together with the fluorescent marker green fluorescent protein GFP.
Experiments were performed as previously described A P value less than 0. RNA-seq experiments were performed on RNA extracted from the tunica muscularis of normal human stomach, a tissue that showed robust Ano1 expression and a diseased state associated with altered expression of Ano1 isoforms By use of a whole-transcriptome sequencing approach, a new exon was identified upstream of the published exon 1.
The aalj sequence had an ATG Fig. The sequencing results from the RACE experiment also demonstrated that the transcriptional start site TSS for the novel isoform is located nucleotides upstream from the translation start site, and it corresponds to the TSS predicted by the Neural Network software.
These data confirm the presence of a previously unidentified exon located 93 kb upstream of the published exon 1 of the human ANO1 gene. This sequence does not contain any previously described protein domains. Identification of a new exon for human ANO1 upstream of the published exon 1. Localization of human ANO1 gene on chromosome 11 and the coverage graphs. C RACE results showing the sequence of the newly described exon 0 green upstream of exon 1 graywith the ATG in exon 0 green, underlined in frame with the ATG in exon 1 gray, underlined.
D Multiple sequence alignment of the human, mouse, cow, and rat Ano1 protein N-terminal region. Prediction of possible promoter regions for the human ANO1 gene was performed using a bp sequence upstream of exon 0. ProScan predicted a putative promoter region between and 40 bp upstream of the previously published TSS, with a score of Both analyses indicate that this promoter may be TATA-less.
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Based on these data, bp of genomic DNA upstream of exon 0 referred to as P0 were amplified by PCR, cloned into the luciferase reporter vector, and transiently transfected into HEK cells.
Twenty-four hours after transfection P0 resulted in a 6. Reporter gene assays showed that the deletion constructs resulted in a significant increase in promoter activity up to Characterization of putative promoter activity. A Bar graph showing the results of the activity analysis by reporter gene luciferase assays of the construct bearing the putative promoter P0.
The results show a 6. Schematic representation of the promoter-reporter constructs containing the promoter fragments of different lengths cloned into the reporter vector. Confirmation of the presence of a transcript containing exon 0 came from quantitative RT-PCR experiments on RNA extracted from T84 cells showing detection of exon 0 by cycle The minimal promoter sequence presented an elevated GC content Methylation analysis was carried out from DNA extracted from muscle strips isolated from human stomach.
As shown in Fig. This is consistent with expression of Ano1 in a subset of cells within the muscle layer. Therefore, P0 is an active and functional promoter for ANO1 with multiple regulatory elements. PolII binds to the minimal P0 promoter region and drives transcription of Ano1. A Sequence of the minimal functional P0 promoter. Putative core promoter elements identified by GPMiner are indicated by boxes.
IL-4 has been shown to positively regulate expression of Ano1 1. In these conditions, the promoter activity of P0 was significantly increased compared with untreated controls Fig. The consensus sites for transcription factors identified in this region of P0 are indicated in Table 5.
The most likely candidate was STAT6, a transcription factor whose role as a downstream effector of human IL-4 signaling has been extensively studied see elsewhere for reviews 37 To test the role of those transcription factors in the up-regulation of Ano1 in response to IL-4, the sequence of the putative binding site on the P0 promoter vector for each of them was disrupted by site-directed mutagenesis.
The results show that the reporter vector fails to respond to IL-4 with an increased activity only in the case of the disruption of the STAT6 putative binding site Fig. Under basal conditions, the activity of each of the mutated construct was not different from the wild-type P0 construct data not shown.
Treatment with IL-4 increased recruitment of STAT6 on the region of the promoter previously identified as the possible binding site Fig. P0 promoter activity is up-regulated by IL A Treatment with IL-4 up-regulated the activity of P0 promoter by 1.
Putative binding sites for transcription factors identified on P0 promoter sequence using GPMiner. A Luciferase assay performed with reporter constructs for P0 where the indicated putative transcription factor binding sites were deleted by site-directed mutagenesis. The top sequence is from the P0 region with the binding sites for transcription factors of interested underlined.
The bar graph shows that only the disruption of the STAT6 binding site rendered the vector unresponsive to IL-4 stimulation. Cotransfection of the P0 reporter vector with an expression vector for STAT6 significantly increases the activity of the promoter by 2.
The results are normalized to a control performed by cotransfection with the empty vector used for STAT6 construct. To test whether the addition of 40 amino acids to the N terminus of Ano1 due to the translation of the exon 0 sequence changes the function of the channel, we examined the current density of the 2 isoforms by whole-cell electrophysiology.
The novel Ano1 isoform results in greater current density. In the present work, we report on a novel promoter region for ANO1.