Abstract P160: A Genome-spanning Map of Chromatin Structure Near the Renin Proximal Promoter

Abstract

Chromatin conformation capture technologies (3C, 4C-seq) allow mapping of the three dimensional spatial structure of the genome. Physical proximity mapping, which captures these 3 dimensional interactions, fills an important gap in our understanding of enhancer-gene relationship by linking possible regulatory regions to specific genes that may be distant from the enhancer by thousands or millions of base pairs or even located on different chromosomes. The goal of the current study was to expand our understanding of renin regulation by identifying regions containing potential regulatory elements of renin more than a few kilobases from the renin proximal promoter, using 4C-seq technology, in isolated SS/JrHrdMcwi cardiac microvascular endothelial cells. Three replicates of ten million cells were fixed, digested with EcoRI, ligated, and then decrosslinked. Secondary digestion with NlaIII followed by a second ligation captured an unknown sequence surrounded by the sequence of the renin promoter. A sequencing library was prepared using primers specific to the renin promoter to identify the captured sequences. Sequence reads were mapped to rat genome (Rn 5) divided into fragments cut by EcoRI. Reads were verified to contain the renin proximal promoter sequence and a uniquely mapped captured sequence. The read counts were binarized and Z-scores were calculated based on a windowed average reads relative to background, and Z-scores corresponding to FDR < 0.01 were considered significant. We found 62 loci spanning the genome in contact with the renin proximal promoter. Clusters of interactions were found on Chr13 at 39.6-40.5Mpb and at 53.9-56.3Mbp where the Renin gene is located. Additional contacting loci were found on all chromosomes except X. Quantitative PCR in newly isolated endothelial cells processed as 3C samples (fixed, digested, ligated, decrosslinked) were used to test 9 of the interactions, of which 8 were validated. These loci are enriched with genes whose expression is correlated with renin (53 of 268 genes, p = 7x10 -10 ) in humans, rats and mice. The present study generated a genome-wide map of segments physically interacting with the renin proximal promoter producing the first such map for a gene that is essential for cardiovascular physiology.