Nuclear factor E2 p45-related factor 2 (symbol Nfe2l2; commonly called Nrf2), a basic-region leucine zipper transcription factor (TF), binds to the cis
-regulatory, antioxidant response element (ARE) and transcriptionally upregulates an environmental stress response gene battery (1
). The Nrf2 response is critical for protection against pulmonary inflammation and emphysema, asthma, acute lung injury, hyperoxia, pulmonary fibrosis, innate immune response, hepatotoxicity, carcinogenesis, neurodegeneration, cardiovascular disease and aging (2–11
). A decline in the Nrf2 pathway is associated with severe chronic obstructive pulmonary disease (COPD) (12–15
). In addition, several classes of small molecules such as 1,2-dithiolethiones (e.g. oltipraz), isothiocyanates (e.g. sulforaphane) and triterpenoids (e.g. CDDO-Im) induce expression of Nrf2 target genes and protect against chemical carcinogenesis, acute hepatotoxicity and cigarette smoke (CS)-induced emphysema (16–19
). On the other hand, studies show that many cancers such as non small cell (NSCLC) and squamous cell lung tumors develop inactivating mutations in the Keap1
genes, respectively, thereby constitutively enhancing the Nrf2 pathway and promoting tumorigenecity and resistance to an array of chemotherapeutic compounds (20
). Hence, due to the dual role of Nrf2 in carcinogenesis and degenerative chronic diseases and the diversity of target genes under a variety of stresses, understanding the pathway components and regulators is critical for effectively targeting the pathway for prophylactic and therapeutic purposes.
Gene-expression profiling studies using different tissue and cell-culture systems exposed to diverse conditions (chemical or environmental) reveal the pleiotropic properties of Nrf2 in stress response and cell survival. Keap1 (Kelch ECH associating protein 1), a cytosolic repressor of the Nrf2 pathway, plays a central role in regulation of the Nrf2 response. Under normal conditions, Nrf2 is targeted by Keap1, which promotes Nrf2 proteasomal degradation via interactions with an ubiquitin ligase (22
). Keap1 further functions as a sensor of stress signals, through stress-induced oxidation of key cysteine residues that lead to conformational changes and the inability to bind Nrf2 (23
). Nrf2 then accumulates in the nucleus, where it binds to AREs in a heterodimeric complex with one of a subset of the small Maf-family of TFs (24
) murine models in different organs and with various stressors, Nrf2 was found to coordinately regulate a suite of targets genes such as antioxidant genes, xenobiotic-metabolizing enzymes, many of which have been traditionally classified as part of the phase II detoxification system, glutathione homeostasis, solute channels, proteome maintenance and innate immune response (8
). Disruption of Keap1
) leads to enhanced nuclear accumulation of Nrf2 and elevated expression of Nrf2-regulated genes (33
). Largely, the Nrf2 regulated genes are associated with pathways related to cell survival—batteries of proteins that provide protection against a variety of oxidative and electrophilic stressors.
Although, many genes are regulated in an Nrf2-dependent manner, no large-scale studies have distinguished between direct and indirect targets of Nrf2, the latter perhaps emerging from Nrf2 cross-talk with other signaling pathways (34–36
). In this report, we evaluated the concordance between global mRNA expression profiles at the transcriptomic levels (i.e., oligonucleotide microarray analyses) and genome-wide Nrf2-DNA-binding analysis using high-throughput methodologies (ChIP-Seq). This approach is important for establishing a systems level view of Nrf2-dependent processes occurring within cells. In the ChIP-Seq analysis, over 1200 Nrf2-bound regions were observed from Keap1−/−
MEFs. These regions were strongly enriched for Nrf2-binding site patterns, with the motifs clustered near the maxima of the ChIP-Seq peaks. The gene-expression analysis identified 7155 genes significantly downregulated in Nrf2−/−
compared with wild-type (WT) MEFs (basal targets) and 7832 significantly upregulated genes in Keap1−/−
compared with WT MEFs (inducible targets), with 2489 modulated genes intersecting between the two datasets (common targets). Integrating the results from ChIP-Seq and expression profiling data, signatures were observed for gene groups that include detoxification response, cell proliferation, cell cycle and survival regulation networks. Strikingly, the proliferation and stress responses segregate between the direct Nrf2 target expression classes, with proliferation linked to basal expression and detoxification linked to inducibleactivation. The high-throughput results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and site-specific ChIP assays in MEFs and CS-exposed mouse lungs to demonstrate the Nrf2 response in vivo
. The integrated analysis of the experimental data highlights the broad influence of Nrf2, providing a blueprint for the regulation of cytoprotective genes and genes involved in critical homeostatic function such as cell proliferation.