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  • br To test whether the


    To test whether the new BACH1-binding sequences partici-pate in Hk2 and Gapdh transcriptional activation, we cloned 400- to 500-bp promoter regions containing the wild-type BACH1 binding sequence, or a mutated version that would pre-vent BACH1 binding, into luciferase reporter vectors (Figure 4E). Luciferase activity from the Hk2 and Gapdh promoters was 4- to 5-fold higher in mTN than in mTC 12-O-tetradecanoyl phorbol-13-acetate (Figures 4H and 4I). Knockout of Bach1 essentially abolished the signal in mTN cells, as did expressing the vectors with mutated sequences (Figures 4H and 4I). Thus, BACH1 stimulates transcription of Hk2 and Gapdh, which raises the possibility that BACH1 regulates glycolysis.
    Antioxidants Stimulate Glycolysis in a BACH1-Dependent Fashion
    Glycolysis rates were 50% higher in mTN than in mTC cells, as judged by analyses of the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR); the OCR/ECAR ratio was reduced correspondingly (Figures 5A, 5B, and S6A). This was accompanied by increased glucose uptake and lactate secretion (Figure 5C). The involvement of glycolysis prompted further analyses of the RNA-seq data. In addition to Hk2 and Gapdh, mTN cells had increased transcript levels of other glyco-lytic enzymes, including 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3) and solute-carrier family 16 member 1 (Slc16a1, encoding the lactate transporter MCT-1) (Figure 5D). Furthermore, analyses of the SEEK co-expression database revealed that in 173 human lung cancer datasets BACH1 expres-sion correlated with HK2 expression (Figure 5E) and tended to correlate with PFKFB3 and SLC16A1 (Figure 5E).
    We next determined whether BACH1 is functionally involved in the increased glycolysis. Using the CRISPR-SAM approach, we
    (E) Schematic showing intratracheal administration of pSECC lentiviruses encoding Cre recombinase, CAS9, and the gRNAs sgTom or sgBach1.
    (F) Metastasis incidence in KP mice 8 months after intratracheal administration of pSECC-sgTom or -sgBach1 lentiviruses; vitamin E (VitE, 0.5 mg/kg) was administered in the chow diet 1 week after the lentiviral infection.
    (G) Schematic of the CRISPR/sgBach1-SAM strategy. sgRNAs target a CAS9-VP64 fusion to the Bach1 promoter, stimulating transcription of the endoge-nous gene.
    (H) Left, western blots showing amounts of BACH1 in mTC cells transduced with SAM-sgBach1; control cells received a nontargeting construct (SAM-sgTom).
    H3 and ACTIN were loading controls. Right, amounts of BACH1 determined by densitometry in two experiments.
    (I) Transwell migration of mTC-SAM-sgBach1 and mTC-SAM-sgTom cells. Right, representative photos of migrated cells.
    (J) Transwell invasion assay of mTC-SAM-sgBach1 and mTC-SAM-sgTom cells.
    Figure 4. Combined Genome Occupancy and Transcriptomic Analyses Identify Hk2 and Gadph as BACH1 Target Genes
    (A) Left, Venn-diagram showing overlap of 240 genes bound by BACH1 in ChIP-seq analyses (green) and genes regulated in RNA-seq analyses (blue). Right,
    STRING analysis of protein-protein interactions (PPI) identified three clusters among the 240 genes: one centered around metabolic processes (circled), one around RHO proteins, and a MYC-centered network. PPI enrichment, p = 4.31e–06.
    (B) Plot of the 20 ‘‘metabolic process’’ genes. x axis, gene expression from RNA-seq data; y axis, level of BACH1 binding identified in the ChIP-seq data.
    (C) qPCR analyses of RNA from mTC and mTN cells (n = 3). Values were normalized to Rplp0 expression and then to mTC.
    (D) Top, western blots showing amounts of HK2 and GAPDH in mTC and mTN cells. Bottom, protein amounts determined by densitometry data from 3 and 1 experiments. respectively.
    (E) Left, identification of BACH1-binding sites 300 bp upstream of transcriptional start sites (TSSs) of Hk2 and Gapdh. Red arrows show primers used for ChIP-qPCR. Right, 400–500 bp of promoter sequences containing the wild-type BACH1 motifs (blue) or mutated motifs (red) were cloned into the pGL3 luciferase reporter vector.