Variability in lung disease severity in cystic fibrosis (CF) is a significant clinical problem. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause CF, a devastating, recessive, monogenic disease. CF is characterized by abnormal ion transport, reduced mucociliary clearance, chronic bacterial infection, inflammatory airway disease with mucus hyper/metaplasia, respiratory failure, and early death. However, the variability in lung phenotype is profoundly influenced by genetic factors that lie outside the CFTR locus. Recent genome-wide association studies (GWAS), led by co-PIs on this proposal, identified a robust association at chromosome 11p13 with lung disease severity. The genomic region encompassing the single nucleotide polymorphisms (SNPs) with highest p-values is located within a ~219kb intergenic region flanked by the Ets homologous factor 1 (EHF) gene on one side and APAF1-interacting protein (APIP) on the other. EHF encodes a protein that belongs to an Ets transcription factor subfamily characterized by epithelial specific expression. APIP is a negative regulator of hypoxic injury and has anti-apoptotic functions. Moreover, there are other genes of potential relevance to lung function close to this region. Our goal is to determine the mechanism whereby genetic elements at 11p13 influence CF lung disease severity, which likely involves key pathobiological pathways (ion transport, inflammation, and/or mucus metaplasia). Since the critical SNPs lie in non-coding regions of the genome it is probable that they are within or close to cis-acting regulatory elements that control the expression of one or more genes within this genomic region. We will pursue three specific aims addressing the main hypothesis that critical regulatory elements for genes at 11p13 are located at or close to the SNPs with highest p-values associating with CF lung disease severity in the replicated GWAS. Moreover, that by finding which elements interact with each gene promoter in the region we will identify genes that are critical for normal lung biology, determine how their expression is regulated and how naturally occurring polymorphisms influence these processes and alter CF disease progression. Experiments in the first aim will identify the location of critical regulatory elements in the 11p13 genomic interval and determine the physical interactions of these cis-acting elements with individual gene promoters. In the second aim, we will determine the function of the critical cis-regulatory elements in controlling expression of relevant genes at 11p13 and how SNPs may alter their properties. Finally we will elucidate the mechanism whereby lung pathology in CF is modulated by proteins encoded by critical gene(s) in the 11p13 genomic interval. The results will determine the biological basis of variability in CF lung disease severity and provide a valuable paradigm for elucidating the molecular basis of other genetic associations with heart, lung and blood diseases that involve cis-acting regulatory elements in non-coding regions distal to genes.
Many genome-wide studies have identified strong association of certain genes (or regions of the genome) with disease. In most cases, however, the specific mechanism causing disease is not understood. We recently identified non-coding regions of the genome that are robustly associated with severity of lung disease in cystic fibrosis (CF). This project was written in response to the NHLBI FOA Getting from Genes to Function in Lung Disease in order to determine the mechanism of CF lung disease that results from those regions, which likely regulate nearby genes. This project has great relevance to public health, not only for CF, but also for other lung diseases with a genetic basis where critical elements are in non-coding, regulatory regions.
|Browne, James A; Harris, Ann; Leir, Shih-Hsing (2014) An optimized protocol for isolating primary epithelial cell chromatin for ChIP. PLoS One 9:e100099|