Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks

Abstract

NF-E2-related factor 2 (Nrf2) is a key transcription factor that is critical for cellular defense against oxidative and xenobiotic insults. Nrf2 heterodimerizes with small Maf (sMaf) proteins and binds to antioxidant response elements (AREs) to activate a battery of cytoprotective genes. However, it remains unclear to what extent the Nrf2-sMaf heterodimers contribute to ARE-dependent gene regulation on a genome-wide scale. We performed chromatin immunoprecipitation coupled with high-throughput sequencing and identified the binding sites of Nrf2 and MafG throughout the genome. Compared to sites occupied by Nrf2 alone, many sites co-occupied by Nrf2 and MafG exhibit high enrichment and are located in species-conserved genomic regions. The ARE motifs were significantly enriched among the recovered Nrf2-MafG-binding sites but not among the Nrf2-binding sites that did not display MafG binding. The majority of the Nrf2-regulated cytoprotective genes were found in the vicinity of Nrf2-MafG-binding sites. Additionally, sequences that regulate glucose metabolism and several amino acid transporters were identified as Nrf2-MafG target genes, suggesting diverse roles for the Nrf2-MafG heterodimer in stress response. These data clearly support the notion that Nrf2-sMaf heterodimers are complexes that regulate batteries of genes involved in various aspects of cytoprotective and metabolic functions through associated AREs.

Figure 1.

Identification of Nrf2- and MafG-binding sites by ChIP-seq analysis. (A) Immunoblot analysis of Nrf2 protein in nuclear lysates of Hepa1 cells treated with 100 -µM DEM or DMSO for 4 h. Lamin B was detected as a loading control. The molecular weight in kDa is shown at right. (B) Validation of the ChIP-seq library. ChIP was performed with Nrf2 and MafG antibodies, and the precipitated DNA was used to make the ChIP-seq library. qPCR was performed to verify the enrichment of regulatory regions of the Nqo1 gene and Hmox-1 gene E1 and E2 enhancers. The third intron of the thromboxane synthase (Txs) gene was used as a negative control locus, and its level was set to 1. (C) Venn diagram showing the overlap between the Nrf2 and MafG-binding sites. Overlapping peaks are defined by an intersection of the distance between the peak summit within 268 bp. (D) The ChIP-seq profiles around the Nqo1 promoter and Hmox-1 E1 and E2 enhancers are shown as UCSC genome browser shots. (E) Identification of the genomic location of Nrf2 single binding sites, Nrf2–MafG-binding sites and MafG single binding sites using CEAS. The diagram illustrates the overall distribution of the Nrf2–MafG-binding sites into the proximal promoters (<–1 kb), distal promoters (–1 to –10 kb), exons, introns and intergenic regions.

Figure 2.

Characterization of TSS-proximal Nrf2- and MafG-binding sites. (A) Venn diagram showing the overlap between the Nrf2- and MafG-binding sites near TSS (±10 kb). (B) Averaged conservation profiles using PhastCons scores for Nrf2–MafG and Nrf2 single binding sites. The profiles of 3.0-kb regions centered on the peak summit are shown. (C) Average profiles of the Nrf2 ChIP-seq signal at the Nrf2–MafG and Nrf2 single binding sites near TSS. The profiles of 2.0-kb regions centered on the peak summit are shown. (D) Distribution of the fold-enrichment values of Nrf2 in the Nrf2–MafG and Nrf2 single binding sites. Classification of the fold-enrichment value is as indicated.

Figure 3.

In vivo Nrf2- and MafG-binding motifs. (A) The consensus sequences of the MARE, original ARE, core ARE and functional ARE (S = G or C, M = A or C, R = A or G, W = A or T). GC boxes are shown in red. The left side and right side of the core ARE are shown to be recognized by Nrf2 and sMaf, respectively. (B, C) The enriched motif identified by the de novo motif-discovery algorithm MEME–ChIP in Nrf2–MafG-binding sites (B) and MafG single binding sites (C). The most significantly enriched motifs are shown. Nucleotide usage at N3 and N13 positions (indicated by arrows) of the enriched ARE is shown (B). (D) Pie charts show the percentage of classified AREs based on the number of variant nucleotides in NNN (from no variant to three variant nucleotides) in the Nrf2–MafG and Nrf2 single binding sites. The ARE motif was searched in the ± 150-nt region centered at the peaks. (E) Nucleotide usage at N8, N9 and N10 positions of the ARE with one variant nucleotide found in the Nrf2–MafG-binding sites. (F) A Pie chart shows the percentage of the TMA-containing ARE in the total core ARE found in the Nrf2–MafG-binding sites. Average profiles of Nrf2 and MafG ChIP-seq signals in the Nrf2–MafG-binding sites with (red line) or without (gray line) TMA motif. The profiles of 2.0-kb regions centered on the peak summit are shown.

Figure 4.

DEM-inducible genes proximal to Nrf2–MafG and Nrf2 single binding sites. Microarray analysis was performed with RNA isolated from DEM- or DMSO-treated Hepa1 cells for 6 h in duplicate. (A) Venn diagram showing the overlap of genes induced by DEM (≥1.5-fold change) and genes proximal to Nrf2–MafG or Nrf2 single binding sites. (B) Venn diagram showing the overlap of genes repressed by DEM (≥1.5-fold change) and genes proximal to the Nrf2–MafG or Nrf2 single binding sites. (C, D) Heat map of differentially expressed genes proximal to the Nrf2–MafG-binding sites (C) or Nrf2 single binding sites (D). DEM-induced genes proximal to Nrf2–MafG-binding sites are categorized into functional groups: antioxidant and detoxification enzymes, proteasome and chaperone, transporter, metabolism and others. The colors of the heat map reflect the log₍₂₎-fold-change values relative to the mean expression level of each gene in the DMSO-treated (Veh) Hepa1 cells. The gene symbols used here are consistent with those used in the Mouse Genome Informatics database.

Figure 5.

Nrf2–MafG heterodimer regulates the expression of NADPH-generating enzyme genes in response to DEM. (A) Nrf2 knockdown and sMaf deficiency impaired DEM-mediated induction of NADPH-generating enzyme gene expression. Hepa1 cells transfected with Nrf2 siRNA or control (Con) siRNA were treated with DEM or DMSO (Veh) for 6 h (n = 3). F0G0K0 and control (Con) MEFs were treated with DEM or DMSO (Veh) for 12 h (n = 4). The mRNA expression was detected by qPCR. The data represent the mean ± SD with P-values derived from ANOVA with the Bonferroni post hoc test: *P < 0.05, **P < 0.01. (B) The ChIP–qPCR analyses performed with chromatin extracts from Hepa1 cells treated with 100 -µM DEM or DMSO (Veh) for 4 h using specific antibodies (SA) for Nrf2, MafG or CBP. Normal IgG was used as a negative control. The amount of immunoprecipitated DNA was analyzed by qPCR with primers flanking the ARE motif in the Nrf2–MafG-binding sites in the Nqo1, Idh1, Pgd and G6pdx genomic regions. The Txs genomic region was used as a negative control. The data represent the mean ± SD (n = 3) with P-values from Student’s unpaired t-test: *P < 0.05, **P < 0.01.

Figure 6.

Stress-inducible histone acetylation at Nrf2–MafG-binding sites. ChIP-seq peak profiles surrounding the Nqo1, Idh1, Pgd and G6pdx genomic loci are shown as UCSC genome browser shots. The genomic conservation (Cons) and PCR primer positions are indicated. ChIP–qPCR analyses were performed under the same condition in Figure 5B using anti-H3Ac, anti-H4Ac or anti-H3K9Ac antibodies. The amount of immunoprecipitated DNA was analyzed by qPCR with primers flanking the ARE motif in the Nrf2–MafG-binding sites and nearly ± 500 bp of this motif in the Nqo1, Idh1, Pgd and G6pdx genomic regions. The data represent the mean ± SD (n = 3) with P-values from Student’s unpaired t-test: *P < 0.05, **P < 0.01. Fold enrichment for DEM treatment relative to DMSO (Veh).