Systems-level analysis reveals selective regulation of Aqp2 gene expression by vasopressin.

Pablo C Sandoval,J'Neka S Claxton,Fahad Saeed,Jae Wook Lee,Mark A Knepper,Jason D Hoffert

doi:10.1038/srep34863

Abstract

Vasopressin-mediated regulation of renal water excretion is defective in a variety of water balance disorders in humans. It occurs in part through long-term mechanisms that regulate the abundance of the aquaporin-2 water channel in renal collecting duct cells. Here, we use deep DNA sequencing in mouse collecting duct cells to ask whether vasopressin signaling selectively increases Aqp2 gene transcription or whether it triggers a broadly targeted transcriptional network. ChIP-Seq quantification of binding sites for RNA polymerase II was combined with RNA-Seq quantification of transcript abundances to identify genes whose transcription is regulated by vasopressin. (View curated dataset at https://helixweb.nih.gov/ESBL/Database/Vasopressin/). The analysis revealed only 35 vasopressin-regulated genes (of 3659) including Aqp2. Increases in RNA polymerase II binding and mRNA abundances for Aqp2 far outstripped corresponding measurements for all other genes, consistent with the conclusion that vasopressin-mediated transcriptional regulation is highly selective for Aqp2. Despite the overall selectivity of the net transcriptional response, vasopressin treatment was associated with increased RNA polymerase II binding to the promoter proximal region of a majority of expressed genes, suggesting a nearly global positive regulation of transcriptional initiation with transcriptional pausing. Thus, the overall net selectivity appears to be a result of selective control of transcriptional elongation.

Highlights

Prior studies have demonstrated that vasopressin increases the steady-state half-life of the aquaporin-2 protein[11,12], but the increase in half-life from 9 to 14 hours is not sufficient to explain the 10-fold or more increase in aquaporin-2 protein normally seen in response to vasopressin[11]
When the reads mapped to Aqp[2] are normalized by the total number of reads for all genes, the log2(dDAVP/Vehicle) value for all replicates was 4.52 ± 0.85, consistent with the prior conclusion that vasopressin markedly increases aquaporin-2 mRNA levels in mpkCCD cells[4,19]
We use the methods of systems biology to investigate mechanisms involved in the long-term regulation of the abundance of the water channel aquaporin-2 by the peptide hormone vasopressin

Summary

Introduction

Prior studies have demonstrated that vasopressin increases the steady-state half-life of the aquaporin-2 protein[11,12], but the increase in half-life from 9 to 14 hours is not sufficient to explain the 10-fold or more increase in aquaporin-2 protein normally seen in response to vasopressin[11]. Of only 35 genes with coincident changes in RNA polymerase II binding and mRNA levels in response to vasopressin, the increases for Aqp[2] were by far the greatest, indicating a highly selective effect of vasopressin signaling on Aqp[2] gene transcription. The major focus of this paper is the regulation of the Aqp[2] gene, an important by-product of the work is a comprehensive listing of mRNA abundances and RNA polymerase II occupancy for genes expressed in cultured mpkCCD cells. This information is provided to users via a publically accessible web page

Methods

Results

Conclusion