Figure 4. MOTS-c Binds to Nuclear DNA and Interacts with Nrf2 to Regulate Gene Expression.
(A and B) Immunoblots of endogenous MOTS-c from chromatin extracts of (A) resting HEK293 cells and (B) HEK293 and HepG2 cells with and without GR (3 hr).
(C) Co-immunoprecipitation of NRF2 by anti-MOTS-c antibody from HEK293 cell nuclear extracts at 0, 3, and 24 hr after GR (top panel) or tBHP (100 μM; bottom panel) determined by immunoblotting.
(D) Co-immunoprecipitation of EGFP-MOTS-c and Myc-tagged NRF2 (Myc- NRF2) in HEK293 cells determined by immunoblotting.
(E) MOTS-cYIFY: 8YIFY11 → 8AAAA11 and pMOTS-cRKLR : 13RKLR16 → 13AAAA16 mutant MOTS-c peptides. Direct MOTS-c/DNA interactions were examined by electrophoretic mobility shift assay (EMSA).
(F) ChIP-qPCR analysis of ARE-containing promoter regions of NQO1 (left) and HO-1 (right) bound to MOTS-c at 0, 3, and 24 hr after GR (top panels) and tBHP (100 μM; bottom panels) (n = 3).
(G) ChIP-qPCR analysis of ARE-containing promoter regions of HO-1 bound to NRF2 in HEK293 cells transfected with empty vector (pEV) or MOTS-c (pMOTS-c) plasmid (n = 3).
(H) ARE-luciferase reporter activity on cells transfected with pEV or pMOTS-c in HEK293 cells in combination with siRNA against NRF2 or a non-specific control (NS) (n = 15).
(I) qRT-PCR analysis of HO-1 and NQO1 expression in response to the overexpression of WT or mutant MOTS-c in HEK293 cells (n = 3).
(J and K) HEK293 cells were stably transfected with pEV, pMOTS-cWT, pMOTS-cYIFY, and pMOTS-cRKLR and subjected to 96 hr of GR + SD. Survival was assessed by (J) phase contrast microscopy images and (K) flow cytometry (n = 3).
(L–N) RNA-seq analyses on HEK293 cells that were transfected with MOTS-c (or empty vector) and subjected to glucose restriction (GR) for 3 hr (n = 6).
(L) Heatmap of significantly differentially regulated genes by MOTS-c upon GR at false discovery rate (FDR) < 5%.
(M) Overlap of genes upregulated by MOTS-c during GR and bona fide NRF2 target genes.
(N) Significantly enriched transcription factor DNA binding motifs in the promoters of genes upregulated (top) and downregulated (bottom) by MOTS-c.
(O) Co-immunoprecipitation of MOTS-c and ATF1 from nuclear extracts of HEK293 cells after GR (3 hr) determined by immunoblotting.
(P) A schematic illustration of the nuclear role of the mitochondrial-encoded peptide MOTS-c in response to metabolic stress.
Error bars represent mean ± SEM. **p < 0.01, ***p < 0.001 by Student’s t test. See also Figure S4 and Tables S1 and S2.