The Mediator Subunit MED16 Transduces NRF2-Activating Signals into Antioxidant Gene Expression

Abstract

The KEAP1-NRF2 system plays a central role in cytoprotection. NRF2 is stabilized in response to electrophiles and activates transcription of antioxidant genes. Although robust induction of NRF2 target genes confers resistance to oxidative insults, how NRF2 triggers transcriptional activation after binding to DNA has not been elucidated. To decipher the molecular mechanisms underlying NRF2-dependent transcriptional activation, we purified the NRF2 nuclear protein complex and identified the Mediator subunits as NRF2 cofactors. Among them, MED16 directly associated with NRF2. Disruption of Med16 significantly attenuated the electrophile-induced expression of NRF2 target genes but did not affect hypoxia-induced gene expression, suggesting a specific requirement for MED16 in NRF2-dependent transcription. Importantly, we found that 75% of NRF2-activated genes exhibited blunted inductions by electrophiles in Med16-deficient cells compared to wild-type cells, which strongly argues that MED16 is a major contributor supporting NRF2-dependent transcriptional activation. NRF2-dependent phosphorylation of the RNA polymerase II C-terminal domain was absent in Med16-deficient cells, suggesting that MED16 serves as a conduit to transmit NRF2-activating signals to RNA polymerase II. MED16 indeed turned out to be essential for cytoprotection against oxidative insults. Thus, the KEAP1-NRF2-MED16 axis has emerged as a new regulatory pathway mediating the antioxidant response through the robust activation of NRF2 target genes.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00785-15.

ACKNOWLEDGMENTS

We thank Keiko Taguchi for providing experimental materials, Masanobu Morita for advice on the CRISPR/CAS9 system, Koichiro Kato and Tatsuro Iso for technical support for recombinant protein preparation, Kyoko Ochiai for advice on protein purification, and the Biomedical Research Core of the Tohoku University Graduate School of Medicine for technical support.

We have no competing interests to declare.

H. Sekine designed the study, conducted the experiments, analyzed the data, and wrote early drafts of the paper. K. Okazaki, N. Ota, H. Shima, and Y. Katoh conducted the experiments and analyzed the data. N. Suzuki, K. Igarashi, and M. Ito contributed to the data analysis. H. Motohashi supervised the research, analyzed the data, and wrote early drafts of the paper. M. Yamamoto supervised the research and wrote the manuscript.

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Funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.