Epigenetic repression of rs6651252 reduces <i>MYC</i> gene expression.

(A). Diagram of a luciferase reporter vector containing a 2.7-kb DNA fragment encompassing rs6651252. The vertical lines above depict the positions of multiple guide RNAs designed to target the element. These guide RNAs recruit the dCas9-KRAB fusion protein to rs6651252 in transfected cells. (B). Luciferase reporter assays conducted in HEK293T cells. Where indicated, plasmids encoding β-catenin S45F, LEF, dCas9-KRAB and plasmids expressing the guide RNAs (gRNAs) were co-transfected. Data are normalized to luciferase levels in cells receiving the reporter vector alone. (C). Relative luciferase reporter activity in HCT116 cells transfected with the plasmids indicated. (D). qPCR analysis of DNA fragments precipitated with a α-FLAG antibody in ChIP assays conducted in HEK293 cells expressing gRNAs alone or gRNAs and FLAG-dCas9-KRAB. The oligonucleotides used for detection amplified a region encompassing rs6651252. (E). RT-qPCR analysis of MYC transcript levels in HCT116 cells expressing gRNAs and the dCas9-KRAB fusion. Non-transfected cells served as a control. Values are normalized to TUBULIN. In (B-E), error bars are SEM (*P P P <0.001).