Abstract P168: ChIP-seq Analysis of Genomic Binding Sites for the Transcription Factor Fosl2 in Kidney Collecting Duct Cells

Abstract

Kidney collecting duct principal cells play a key role in blood pressure regulation and water balance in part through the action of vasopressin. We integrated data from prior proteomics and transcriptomics studies of cultured mouse mpkCCD cells using Bayes’ Rule to rank transcription factors (TFs) likely to be involved in vasopressin-mediated transcriptional regulation. The top ranked TF was the AP-1 component Fos-like 2 (Fosl2) and the third ranked TF was Sox4. We carried out ChIP-seq analysis (HiSeq 2000 sequencer) to identify genomic sites of Fosl2 binding using an antibody successfully employed for ChIP-seq in the Mouse ENCODE Project. mpkCCD cells treated with the vasopressin analog dDAVP or vehicle were separately analyzed. The success of the immunoprecipitation of Fosl2 was confirmed by western blotting and LC-MS/MS of the immuno-precipitated material (ChIP-MS). ChIP-MS identified two different Fosl2 peptides, but there were no peptides corresponding to other AP-1 proteins. ChIP-MS also identified Bub1b, a nuclear protein kinase known to phosphorylate histones, as a Fosl2-interacting protein. Fosl2-binding sites successfully mapped to genes by ChIP-seq included a Fosl2 binding site, centered at 87 bp upstream from the transcription start site [TSS] of Sox4, that was strongly downregulated in response to dDAVP, n=2; confirmed by ChIP-PCR. TF binding site prediction (Genomatix) identifies putative Sox4 binding sites in vasopressin-regulated genes beta-ENaC (-822 bp from TSS) and aquaporin-2 (-515 bp from TSS). These data provide an initial step in identification of the vasopressin-regulated transcriptional network in renal collecting duct cells.