Document Type
Article
Publication Date
2014
College/Unit
Eberly College of Arts and Sciences
Department/Program/Center
Biochemistry
Abstract
An elevated plasma aldosterone and an increased expression of the intermediate conductance K+ (IK/Kcnn4) channels are linked in colon. This observation suggests that the expression of Kcnn4 gene is controlled through the action of aldosterone on its cognate receptor (i.e., mineralocorticoid receptor; MR). In order to establish this, we performed chromatin immunoprecipitation (ChIP) assay to identify the MR response elements (MREs) in a region that spanned 20 kb upstream and 10 kb downstream of the presumed transcription start site (TSS) using chromatin from the colonic epithelial cells of normal and aldosterone-treated rats. MREs were immunoprecipitated in an approximately 5 kb region that spanned the first and second introns in the aldosterone rats. These regions were individually cloned in luciferase-expression vector lacking enhancer activity. These clones were tested for enhancer activity in vitro by transfecting in HEK293T and CaCo2 cells with MR and aldosterone treatment. At least four regions were found to be responsive to the MR and aldosterone. Two regions were identified to contain MREs using bioinformatics tools. These clones lost their enhancer activity after mutation of the presumptive MREs, and thus, established the functionality of the MREs. The third and fourth clones did not contain any bioinformatically obvious MREs. Further, they lost their activity upon additional sub-cloning, which suggest cooperativity between the regions that were separated upon sub-cloning. These results demonstrate the presence of intronic MREs in Kcnn4 and suggest a highly cooperative interaction between multiple intronic response elements.
Digital Commons Citation
O'Hara, Bryan; Rosa, Diego A.; and Rajendran, Vazhaikkurichi M., "Multiple Mineralocorticoid Response Elements Localized in Different Introns Regulate Intermediate Conductance K+ (Kcnn4) Channel Expression in the Rat Distal Colon" (2014). Faculty & Staff Scholarship. 2552.
https://researchrepository.wvu.edu/faculty_publications/2552
Source Citation
O’Hara B, de la Rosa DA, Rajendran VM (2014) Multiple Mineralocorticoid Response Elements Localized in Different Introns Regulate Intermediate Conductance K+ (Kcnn4) Channel Expression in the Rat Distal Colon. PLoS ONE 9(6): e98695. https://doi.org/10.1371/journal.pone.0098695
Comments
© 2014 O’Hara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.