Beyond antioxidant genes in the ancient Nrf2 regulatory network

https://doi.org/10.1016/j.freeradbiomed.2015.06.044Get rights and content

Highlights

  • The Nrf2 regulatory network is strongly conserved from Drosophila to human.

  • Deeply conserved Nrf2 transcriptional targets include antioxidant genes, proteasomal genes, glucose and lipid metabolism genes, Keap1, and more.

  • Nrf2 employs multiple regulatory strategies for transcriptional activation of its target genes.

Abstract

Nrf2, a basic leucine zipper transcription factor encoded by the gene NFE2L2, is a master regulator of the transcriptional response to oxidative stress. Nrf2 is structurally and functionally conserved from insects to humans, and it heterodimerizes with the small MAF transcription factors to bind a consensus DNA sequence (the antioxidant response element, or ARE) and regulate gene expression. We have used genome-wide chromatin immunoprecipitation and gene expression data to identify direct Nrf2 target genes in Drosophila and humans. These data have allowed us to construct the deeply conserved ancient Nrf2 regulatory network—target genes that are conserved from Drosophila to human. The ancient network consists of canonical antioxidant genes, as well as genes related to proteasomal pathways and metabolism and a number of less expected genes. We have also used enhancer reporter assays and electrophoretic mobility-shift assays to confirm Nrf2-mediated regulation of ARE activity at a number of these novel target genes. Interestingly, the ancient network also highlights a prominent negative feedback loop; this, combined with the finding that Nrf2-mediated regulatory output is tightly linked to the quality of the ARE it is targeting, suggests that precise regulation of nuclear Nrf2 concentration is necessary to achieve proper quantitative regulation of distinct gene sets. Together, these findings highlight the importance of balance in the Nrf2–ARE pathway and indicate that Nrf2-mediated regulation of xenobiotic metabolism, glucose metabolism, and proteostasis has been central to this pathway since its inception.

Section snippets

Chemicals and materials

d,l-Sulforaphane (SFN) was purchased from MP Biomedicals (Santa Ana, CA, USA) or Calbiochem/Millipore (Billerica, MA, USA) and ethyl-7-chloro-4-hydroxy-8-methylquinoline-3-carboxylate (AI-1) was purchased from Calbiochem/Millipore. Luperox (tert-butylhydroperoxide solution; TBOOH) was purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Stock solutions of all test compounds were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. FuGENE HD transfection reagent was purchased

Overview of Cnc/Nrf2 genome-wide binding in Drosophila

To get a genome-wide view of the Cnc/Nrf2 regulatory network in Drosophila, we performed ChIP-seq using an antibody against the C-terminal region of Cnc/Nrf2 that recognizes all Cnc isoforms (Fig. 1B). Chromatin was isolated from embryos (16–24 h after egg laying), white prepupae (at the transition between larval and pupal stages), or 2- to 3-day-old adults (male or female). Although it is a stress-responsive transcription factor, CncC—the Nrf2 ortholog—also regulates cytoprotective genes under

Conclusion

Together, these data demonstrate that the Keap1–Nrf2–MAF regulatory axis is strongly conserved from D. melanogaster to humans, even at the level of network structure. The regulatory reach of Nrf2 clearly extends well beyond antioxidant genes, covering metabolic genes, proteostasis genes, autoregulatory pathways, and more; this property of the Nrf2 network is also an ancient one. Importantly, because Nrf2 targets a variety of distinct gene batteries, it is also likely that Nrf2 employs more than

Acknowledgments

The authors thank Evan Odean (University of Minnesota), Lijia Ma (University of Chicago), Anshul Kundaje (Stanford), and Katherine Harris (SwitchGear Genomics) for technical assistance. This work was supported by funding from the University of Minnesota Foundation (M.S.) and the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (Z01-ES-100475 and ES065079 to D.A.B.).

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