Integration of a scalable parallel reporter genes for qRT-PCR based enhancer screening with CRISPR dCas9 activation and supershift EMSA identifies impaired BACH1 repression of ST8SIA1 to contribute to the increased risk for periodontitis in smokers

Genome-wide association studies identified many loci associated with human diseases, but assigning causal alleles remains difficult. Likewise, generating biological meaning underlying a statistical association is challenging. To address these problems, we developed a scalable reporter gene system for the simultaneous transfection and parallel quantification of enhancer activity. Additionally, we characterized a genetic association at the gene ST8SIA1 that increases the risk for periodontitis in smokers. We cloned unique DNA barcode sequences to a reporter gene. Transcript levels of the barcodes and firefly luminescence of the reporter was compared in HeLa cells for several known enhancers. We mapped the associated SNPs to DNA elements with predictive features of regulatory function on gene expression from ENCODE. We tested whether associated alleles changed predicted transcription factor binding sites and proved transcription factor binding using antibody electrophoretic mobility shift assays. Using CRISPR dCas9 activation, we pinpointed the target gene of the association. Using RNA-Sequencing, we identified gene sets that are associated with ST8SIA1. Two associated repressor elements bind the transcription factor BACH1 with the putative causative variant rs2012722 decreasing BACH1 binding by 40%. ST8SIA1 is the likely target gene of the association, regulating the cell cycle arrest and integrin cell surface signaling. We found the periodontitis risk gene ABCA1 as the most significantly up-regulated gene. In conclusion, we developed a low cost, easy to use parallel reporter gene system that facilitates enhancer screening, and described an experimental pipeline for straightforward identification and characterization of causal variants and of their target genes. 500-1000 ng of total RNA of the 6 transfected independent HeLa cell cultures was sequenced with 16 million reads (75bp single end) on a NextSeq 500 using the NextSeq 500/550 High Output Kit v2.5 (75 Cycles). RNA-Seq was performed at the Berlin Institute of Health Core Facility Genomics.