Regulatory dynamics of 11p13 suggest a role for EHF in modifying CF lung disease severity

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), but are not good predictors of lung disease phenotype. Genome-wide association studies (GWAS) previously identified additional regions of the genome associated with CF disease severity. One of these, at chromosome 11p13, is an intergenic region between Ets homologous factor (EHF) and Apaf-1 interacting protein (APIP). Our goal was to determine the functional significance of this region, which is predicted to be regulatory, since it lacks annotated genes. To identify cis-regulatory elements within the chr11p13 region in lung epithelial cells, we first mapped open chromatin, using DNase I digestion and deep sequencing (DNase-seq). Next, histone modifications associated with active chromatin, H3K4me1 and H3K27ac, were revealed by chromatin immunoprecipitation. Predicted enhancer elements were assayed in bronchial epithelial cells using luciferase reporter genes. Two elements showed strong enhancer activity for the promoters of both EHF and the 5’ adjacent gene E47 like ETS transcription factor 5 (ELF5). No enhancers of the APIP promoter were found. Next, to determine direct physical interactions between cis-regulatory elements and gene promoters within 11p13 we used circular chromosome conformation capture (4C-seq). 4C-seq confirmed the enhancer-promoter associations, identified additional interacting elements and defined the boundaries of the topologically associated domains (TADs) at 11p13, which are enriched for CTCF. No strong interactions were observed with the APIP promoter, which lies outside of the main TAD encompassing the GWAS signal. These results focus attention on the role of EHF in modifying CF lung disease severity. To identify regulatory elements at the 11p13 cystic fibrosis modifier region, DNase-seq was performed on 16HBE14o- transformed lung bronchial epithelial cells, and ChIP-seq for H3K4me3, H3K27me3, H3K9me2, H3K9me3, and H3K36me3 was performed in Calu3 lung adenocarcinoma cells and primary human bronchial epithelial cells