Article Figures & Data
Figures
- FIG 1
NFE2L1 and NFE2L3 complementarily regulate proteasome activity and proteasome subunit gene expression at the basal level. (A) Impact of NFE2L knockdown on basal proteasome activity. At 24 h after siRNA transfection into ZsPS cells, the fluorescence intensity derived from a ZsProSensor-1 reporter was measured using flow cytometry. The cell populations in Q1 enclosed by a red line are those with low proteasome activity. siNFE2L1/3 represents the double knockdown of NFE2L1 and NFE2L3. Control siRNA (siCont) was used as a control. ***, P < 0.005; n.s., not significant (n = 3, means + SD, ANOVA followed by Tukey’s test). (B) Impact of NFE2L knockdown on BTZ resistance. At 24 h after siRNA transfection into HCT116 cells, the cells were treated with 5 nM BTZ and further incubated for 48 h. The cells then were subjected to cell viability assay using trypan blue staining. siNFE2L1/3 represents the double knockdown of NFE2L1 and NFE2L3. Control siRNA (siCont) and dimethyl sulfoxide (DMSO) were used as controls. Cell viability was determined by the number of living cells with BTZ treatment normalized by that with DMSO. *, P < 0.05; n.s., not significant (n = 3, means + SD, ANOVA followed by Tukey’s test). (C) Impact of NFE2L knockdown on mRNA levels of 17 common core genes with a “yes” value in core enrichment in both NFE2L1 and NFE2L3 knockdown cells (Table S2). At 48 h after siRNA transfection into HCT116 cells, the cells were analyzed by RT-qPCR. siNFE2L1/3 represents double knockdown of NFE2L1 and NFE2L3. Control siRNA (siCont) was used as a control. mRNA levels of each proteasome subunit were normalized according to levels of β-actin mRNA (n = 3, mean + SD, ANOVA followed by Tukey’s test; *, siCont versus siNFE2L1; †, siCont versus siNFE2L3; §, siCont versus siNFE2L1/3). (D) ChIP peaks of NFE2L1 and NFE2L3 in the promoters of proteasome-related genes. ChIP sequencing of endogenous NFE2L1 or exogenous NFE2L3 was performed using wild-type HCT116 cells or NFE2L3-overexpressing H1299 cells treated with 1 μM MG-132 for 24 h.
- FIG 2
NFE2L3 represses NFE2L1 translation by inhibiting polysome formation on NFE2L1 mRNA. (A and B) Impact of NFE2L knockdown on protein and mRNA levels of another NFE2L in HCT116 (colorectal carcinoma) cells. At 48 h after siRNA transfection, the cells were analyzed by immunoblotting (A) and RT-qPCR (B). Protein or mRNA levels of each NFE2L were normalized with reference to α-tubulin protein or β-actin mRNA, respectively. Control siRNA (siCont) was used as a control. *, P < 0.05; n.s., not significant (n = 3, means + SD; t test in panel A, ANOVA followed by Tukey’s test in panel B). (C) Impact of NFE2L knockdown on protein degradation of another NFE2L. At 48 h after siRNA transfection, the cells were treated with CHX and analyzed by immunoblotting at the times indicated. Protein levels were normalized by α-tubulin. Control siRNA (siCont) was used as a control (n = 3, means + SD). (D) Distribution of NFE2L mRNA in HCT116 transfected with the indicated siRNA (n = 2). At 48 h after siRNA transfection, the cells were analyzed by sucrose gradient centrifugation followed by RT-qPCR. mRNA levels in each fraction were normalized against those in the input. Mean and individual values are represented as a line and mark, respectively. Control siRNA (siCont) was used as a control.
- FIG 3
CPEB3 is an NFE2L3 target gene that negatively regulates NFE2L1 translation. (A) Impact of NFE2L3 knockdown on mRNA levels of CPEB3 in HCT116 cells (left) and NFE2L3-overexpressing H1299 cells (right). Control siRNA (siCont) or GFP-overexpressing H1299 cells were used as controls. mRNA levels of CPEB3 were normalized according to β-actin mRNA. *, P < 0.05 (n = 3, means + SD, t test). (B and C) The recruitment of NFE2L3 on CPEB3 promoters in NFE2L3-overexpressing H1299 cells. GFP-overexpressing H1299 cells were used as controls. In panel B, the genome locus of the CPEB3 promoter and multiple sequences of two candidate AREs in the indicated species are shown. TSS, transcription start site. **, P < 0.01; n.s., not significant (n = 3, means + SD, t test). (D) Impact of CPEB3 knockdown on NFE2L1 protein levels. Each siRNA was transfected into HCT116 cells. After 48 h, the cells were analyzed by immunoblotting. Representative images of immunoblotting are shown in the left panel, and the protein levels were normalized by α-tubulin in the right panel. Control siRNA (siCont) was used as a control. *, P < 0.05 (n = 3, means + SD, ANOVA followed by Tukey’s test). (E) Impact of CPEB3 knockdown on the amount of NFE2L1 mRNA on polysomes. At 48 h after siRNA transfection, the cells were subjected to sucrose gradient centrifugation. Fractions 2 to 8 and 11 to 19, shown in Fig. S3D, were collected as the nonpolysomal and polysomal fractions, respectively. Each fraction was analyzed by RT-qPCR. mRNA levels of NFE2L1 in each fraction were normalized against those in the input. NFE2L1 mRNA levels in the polysomal fractions then were divided by NFE2L1 mRNA levels in the nonpolysomal fractions. Control siRNA (siCont) and siNFE2L3 were used as controls. Means + SD and three independent values are represented as bars and indicated colored marks, respectively (n = 3). (F) Impact of CPEB3 overexpression on NFE2L1 protein levels. At 48 h after 3×Flag-CPEB3 plasmid transfection into HCT116 cells, the cells were analyzed by immunoblotting. p3×FLAG-CMV 10 vector without any fusion proteins was used as a control empty vector (Emp). (G) Interactions between CPEB3 protein and NFE2L1 mRNA. At 24 h after 3×Flag-CPEB3 plasmid transfection, cells were analyzed by RIP assay followed by RT-qPCR. RNA immunoprecipitated mRNA levels of NFE2L1 were normalized against the input values. p3×FLAG-CMV 10 vector without any fusion proteins was used as a control empty vector (Emp). *, P < 0.05 (n = 3, means + SD, t test). (H and I) Impact of NFE2L3 or CPEB3 knockdown on NFE2L1 translation via its 3′UTR. At 24 h after the transfection of siNFE2L3 (H) or siCPEB3 (I), a luciferase reporter vector fused with NFE2L1-3′UTR was transfected and cultured for 24 h. Control siRNA (siCont) was used as a control. Luciferase activity was normalized by mRNA levels of a luciferase gene. *, P < 0.05 (n = 3, means + SD, t test). (J) Impact of NFE2L1-3′UTR deletion or mutation on translation. The luciferase reporter vector fused with the indicated NFE2L1-3′UTR was transfected into HCT116 cells, and the cells were analyzed after 24 h. Luciferase activity was normalized according to the mRNA levels of a luciferase gene. Red and blue rectangles represent wild-type (WT) CPEs, which are highlighted in Fig. S3H, and an adenine-mutated CPE#2 (mCPE#2), respectively. **, P < 0.01; n.s., not significant (n = 3, means + SD, ANOVA followed by Tukey’s test).
- FIG 4
NFE2L3 and CPEB3 contribute to maintain proteasome activity in cancer cells and the prognosis of colorectal cancer patients. (A to C) Impact of CPEB3 overexpression under NFE2L3 knockdown on the expression of the proteasome-related genes (A), proteasome activity (B), and BTZ resistance (C). HCT116 or ZsPS cells were used in panels A and C or in panel B, respectively. At 24 h after NFE2L3 siRNA transfection, the cells were additionally transfected with the 3×Flag-CPEB3 plasmid and cultured for 24 h. Control siRNA (siCont) and p3×FLAG-CMV 10 empty vector (Emp) without any fusion proteins were used as controls. In panel A, the cells were subjected to RT-qPCR using the primers for seven proteasome-related genes analyzed in Fig. 1C. mRNA levels of each proteasome subunit were normalized according to the levels of β-actin mRNA. In panel B, the fluorescence intensity derived from a ZsProSensor-1 reporter was measured using flow cytometry. The cell populations in Q1 enclosed by a red line are those with low proteasome activity. In panel C, the cells were treated with 10 nM BTZ and further incubated for 24 h. The cells then were subjected to cell viability assay using trypan blue staining. Cell viability was determined by the number of living cells with BTZ treatment normalized by that with DMSO. *, P < 0.05; **, P < 0.001; ***, P < 0.005; n.s., not significant (n = 3, mean + SD, t test). (D) Kaplan-Meier analysis comparing overall survival between groups expressing higher and lower levels of CPEB3/NFE2L3. The median values for CPEB3 gene expression levels, normalized by NFE2L3 gene expression levels, were used as the thresholds between tumors expressing higher and lower levels of CPEB3/NFE2L3 or expressing higher and lower levels of CPEB3/NFE2L1, respectively. The hazard ratio (HR) was calculated based on Cox’s proportional hazards model. Data are from patients having colorectal adenocarcinoma (COAD) or rectal adenocarcinoma (READ) from The Cancer Genome Atlas. (E) Schematic model of the mechanism of cross talk between NFE2L1 and NFE2L3 to complementarily maintain basal proteasome gene expression and activity in cancer cells through CPEB3-mediated translational repression.
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Fig. S1 to S5 and Tables S1 to S4
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