(R4057) Functional Analysis of the Enhancer at cg05575921, a DNA Methylation Site that is Tobacco Smoke-Responsive and Highly Associated with Lung Cancer Risk

Anastasiya Demenko (USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California)| Chunli Yan (USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California)| Ite Offringa (USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California)

This project aims to investigate the molecular role of epigenetic changes from tobacco smoke exposure in the development of lung cancer, the largest cause of cancer-related deaths. The epigenome consists of the heritable chemical groups on the genome that regulate gene expression. One well-studied epigenetic mark is DNA methylation, which occurs at CpG dinucleotides and is usually associated with gene silencing. Environmental and lifestyle factors such as smoking can induce heritable alterations to the epigenome and can thereby influence disease risk. However, the mechanisms by which tobacco smoke exposure leads to increased lung cancer risk are not fully understood. Past studies have linked hypomethylation of cg05575921, a CpG in the aryl hydrocarbon receptor repressor (AHRR) gene, a regulator of the xenobiotic response pathway, as a strong indicator of lung cancer risk. Tobacco smoke exposure results in hypomethylation of cg05575921 in blood and lung tissue. Our previous research showed that cg05575921 flanks a tobacco smoke-inducible enhancer linked to the induction of AHRR expression. Our guiding hypothesis is that hypomethylation of cg05575921 is a byproduct of enhancer activation by tobacco smoke, and that it is the enhancer activity that has functional consequences, not the loss of methylation at cg05575921. To evaluate the functional role of this enhancer, we used CRISPR/Cas9 gene editing technology to generate monoclonal A549 cell lines with targeted DNA deletions of either the enhancer region, the CpG site adjacent to it, or a negative control region lacking regulatory marks. Resulting clones will be treated with cigarette smoke condensate (CSC) and analyzed for AHRR expression through whole-transcriptome mRNA sequencing (RNA-seq). Initial findings show a high rate of successful deletions in transfected cells, bolstering the technique’s potential for studying enhancers’ roles in cancerous transformation. Thus far, clones in the enhancer deletion group have been screened via PCR for the desired deletion, further characterized via Sanger sequencing across deletion sites and heterozygous SNPs, exposed to CSC for 48 hours, and RNA has been subjected to RNA-seq. Analysis of RNA-seq data is ongoing. CpG deletion clones have been screened and sequenced and are ready to undergo CSC exposure. Negative control deletion clones have been generated and are growing in preparation for screening. Our analyses promise to shed light on the mechanism underlying the link between cg05575921 hypomethylation and lung cancer risk.



Overview of Engineered Deletion Regions. The red line marks the position of cg05575921. Each group of clones edited with CRISPR/Cas9 had 1 of 3 deletions. The gray highlight marks the negative control deletion region. The yellow highlight marks the CpG deletion region. The blue highlight marks the enhancer deletion region. The colored "hills" mark regions bound by histone proteins that carry modifications typical of enhancers. (Retrieved from the UCSC Genome Browser, including ENCODE data.)