Comparison of Hepatic NRF2 and Aryl Hydrocarbon Receptor Binding in 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Treated Mice Demonstrates NRF2-Independent PKM2 Induction.

ABSTRACT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic oxidative stress following activation of the aryl hydrocarbon receptor (AhR). Our recent studies showed TCDD induced pyruvate kinase muscle isoform 2 (Pkm2) as a novel antioxidant response in normal differentiated hepatocytes. To investigate cooperative regulation between nuclear factor, erythroid derived 2, like 2 (Nrf2) and the AhR in the induction of Pkm2, hepatic chromatin immunoprecipitation sequencing (ChIP-seq) analyses were integrated with RNA sequencing (RNA-seq) time-course data from mice treated with TCDD for 2-168 hours. ChIP-seq analysis 2 hours after TCDD treatment identified genome-wide NRF2 enrichment. Approximately 842 NRF2-enriched regions were located in the regulatory region of differentially expressed genes (DEGs), whereas 579 DEGs showed both NRF2 and AhR enrichment. Sequence analysis of regions with overlapping NRF2 and AhR enrichment showed over-representation of either antioxidant or dioxin response elements, although 18 possessed both motifs. NRF2 exhibited negligible enrichment within a closed Pkm chromatin region, whereas the AhR was enriched 29-fold. Furthermore, TCDD induced Pkm2 in primary hepatocytes from wild-type and Nrf2-null mice, indicating NRF2 is not required. Although NRF2 and AhR cooperate to regulate numerous antioxidant gene expression responses, the induction of Pkm2 by TCDD is independent of reactive oxygen species-mediated NRF2 activation.