Identification of an ATRS in the MMP2 promoter. (A) Left, A list of the MMP2 promoter luciferase reporter constructs. The promoter region is indicated relative to the transcription start site. The arrows indicate the 2 primer pairs used for PCR amplification in the ChIP assay. The putative ATRS in the middle region of the MMP2 promoter is indicated. Right, 293T cells transfected with the 3 pMMP2 Luc constructs with or without pCMV-NLS-EGFR, were subjected to a luciferase reporter gene assay. (B) H1975 cells were analyzed by ChIP using preimmune IgG or anti-EGFR antibody as described in the Materials and Methods. The relative amount of immunoprecipitated DNA from the middle or distal regions of the MMP2 promoter was quantified by RT-qPCR with specific primers. (C) Upper, The sequences of the wild-type and mutant pMMP2 Luc near the ATRS are provided. The wild-type and mutated ATRS sequences are underlined with the mutated nucleotides in boldface. Lower, 293T cells transfected with wild-type or mutant ATRS pMMP2 Luc with or without pCMV-NLS-EGFR were subjected to a luciferase reporter gene assay. (D) 293T cells transfected with pMMP2 Luc with a wild-type or a mutant ATRS and pCMV-NLS-EGFR were subjected to ChIP with an anti-EGFR antibody. The relative level of immunoprecipitated wild-type or mutant MMP2 ATRS were Figure 3 (See previous page). quantified by RT-qPCR with specific primers. (E) 293T cells transfected with pMMP2 Luc with a wild-type or a mutated ATRS and increasing amounts of pCMV-NLS-EGFR were subjected to ChIP with an anti-H3K9Ac antibody. The relative amount of ATRS derived from the immunoprecipitated MMP2 promoter was quantified by RT-qPCR with specific primers.