Identification of a Functional Antioxidant Response Element within the Eighth Intron of the Human ABCC3 Gene

The ATP-binding cassette (ABC) family of transporters, including ABCC3, is a large family of efflux pumps that plays a pivotal role in the elimination of xenobiotics from the body. ABCC3 has been reported to be induced during hepatic stress conditions and through the progression of some forms of cancer. Several lines of evidence have implicated the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in this induction. However, although rodent models have been investigated, a functional antioxidant response element (ARE) in the human ABCC3 gene has not been identified. The purpose of this study was to identify and characterize the ARE(s) responsible for mediating the Nrf2-dependent induction of the human ABCC3 gene. A high-throughput chromatin immunoprecipitation-sequencing analysis performed in A549 cells revealed a specific interaction between Nrf2 and the eighth intron of the human ABCC3 gene rather than the more prototypical flanking region of the gene. Subsequent in silico analysis of the intron identified two putative ARE elements that contained the core consensus ARE sequence commonly found in several Nrf2-responsive genes. Functional characterization of these two AREs using luciferase-reporter constructs with ARE mutant constructs revealed that one of these putative AREs is functionally active. Finally, DNA pull-down assays confirmed specific binding of these intronic AREs by Nrf2 in vitro. Our findings identify a functional Nrf2 response element within the eighth intron of the ABCC3 gene, which may provide mechanistic insight into the induction of ABCC3 during antioxidant response stimuli.