Background Renal water excretion is controlled by vasopressin, in part through regulation of the transcription of the aquaporin-2 gene (Aqp2). Methods To identify enhancer regions likely to be involved in the regulation of Aqp2 and other principal cell-specific genes, we used several next generation DNA-sequencing techniques in a well characterized cultured cell model of collecting duct principal cells (mpkCCD). To locate enhancers, we performed the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) to identify accessible regions of DNA and integrated the data with data generated by chromatin immunoprecipitation followed by next generation DNA-sequencing (ChIP-Seq) for CCCTC binding factor (CTCF) binding, histone H3 lysine-27 acetylation, and RNA polymerase II. ResultsWeidentified two high-probability enhancers centered 81 kbupstream and 5.8 kbdownstream from the Aqp2 transcriptional start site.Motif analysis of these regions and the Aqp2 promoter identified several potential transcription factorbinding sites, including sites for twob-ZIP transcription factors:CCAAT/enhancer binding protein-b (C/EBPb) and cAMP-responsive element binding protein (CREB). To identify genomic binding sites for both, we conducted ChIP-Seq using well characterized antibodies. In the presence of vasopressin, C/EBPb, a pioneer transcription factor critical to cell-specific gene expression, bound strongly at the identified enhancer downstream from Aqp2. However, over multiple replicates, we found no detectable CREB binding sites within 390 kb of Aqp2. Thus, any role for CREB in the regulation of Aqp2 gene transcription is likely to be indirect. Conclusions The analysis identified two enhancer regions pertinent to transcriptional regulation of the Aqp2 gene and showed C/EBPb (but not CREB) binding.
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