Genome-Wide Mapping of DNA Accessibility and Binding Sites for CREB and C/EBP<i>β</i> in Vasopressin-Sensitive Collecting Duct Cells.

Abstract

<b>Background</b> Renal water excretion is controlled by vasopressin, in part through regulation of the transcription of the aquaporin-2 gene (<i>Aqp2</i>).<b>Methods</b> To identify enhancer regions likely to be involved in the regulation of <i>Aqp2</i> 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.<b>Results</b> We identified two high-probability enhancers centered 81 kb upstream and 5.8 kb downstream from the <i>Aqp2</i> transcriptional start site. Motif analysis of these regions and the <i>Aqp2</i> promoter identified several potential transcription factor binding sites, including sites for two b-ZIP transcription factors: CCAAT/enhancer binding protein-<i>β</i> (C/EBP<i>β</i>) 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/EBP<i>β</i>, a pioneer transcription factor critical to cell-specific gene expression, bound strongly at the identified enhancer downstream from <i>Aqp2</i> However, over multiple replicates, we found no detectable CREB binding sites within 390 kb of <i>Aqp2</i> Thus, any role for CREB in the regulation of <i>Aqp2</i> gene transcription is likely to be indirect.<b>Conclusions</b> The analysis identified two enhancer regions pertinent to transcriptional regulation of the <i>Aqp2</i> gene and showed C/EBP<i>β</i> (but not CREB) binding.