Abstract
Mitochondria contain a 16-kb double stranded DNA genome encoding 13 proteins essential for respiration, but the mechanisms regulating transcription and their potential role in cancer remain elusive. Although methyl-CpG-binding ___domain (MBD) proteins are essential for nuclear transcription, their role in mitochondrial DNA (mtDNA) transcription is unknown. Here we report that the MBD2c splicing variant translocates into mitochondria to mediate mtDNA transcription and increase mitochondrial respiration in triple-negative breast cancer (TNBC) cells. In particular, MBD2c binds the noncoding region in mtDNA and interacts with SIRT3, which in turn deacetylates and activates TFAM, a primary mitochondrial transcription factor, leading to enhanced mtDNA transcription. Furthermore, MBD2c recovered the decreased mitochondrial gene expression caused by the DNA synthesis inhibitor cisplatin, preserving mitochondrial respiration and consequently enhancing drug resistance and proliferation in TNBC cells. These data collectively demonstrate that MBD2c positively regulates mtDNA transcription, thus connecting epigenetic regulation by deacetylation with cancer cell metabolism, suggesting druggable targets to overcome resistance.
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Data availability
The ChIP−seq and BS-seq data have been deposited in the Gene Expression Omnibus (GEO) under the accession codes GSE263933 and GSE270688. The human genome sequence (hg19) downloaded from the UCSC genome browser (https://genome.ucsc.edu/) was used as the reference for mapping. Proteome Discovery version 1.3 using the MASCOT search engine was used to analyze protein acetylation data. All other data are available in Supplementary Information that are provided with this paper. Source data are provided with this paper.
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Acknowledgements
This work is supported in part by the National Natural Science Foundation of China (82192893, 81930083 and 81821001 to H.Z., 82130087, 92357301 and 82341013 to P.G., 82422057 and 82273221 to L.S.), the Chinese Academy of Sciences (XDB0940101 to H.Z.), the National Key R&D Program of China (2022YFA1304504 to H.Z.), the Hefei Comprehensive National Science Center Institute of Health and Medicine Project (DJK-LX-2022001 to H.Z.), the Basic Funding of Guangzhou Municipal Science and Technology Bureau (2024A04J6493 to L.S.) and the Special Program for Young Scholars of Southern Medical University (G623281133 to L.S.).
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P.G. and H.Z. conceived the study and supervised experiments. Y.H., Z.Z., L.S., P.G. and H.Z. designed experiments. Y.H., Z.Z., R.L., H.L., Y.Z., Y.S., Q.M., T.Z., S.-T.L., Z.L. and Y.C. performed experiments. Y.H. and S.S. analyzed ChIP−seq data. Y.H., L.S., P.G. and H.Z. wrote the paper. All authors read and approved the paper.
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Extended data
Extended Data Fig. 1 MBD2c trans-localizes into mitochondria via TOM20 and binds to the noncoding region.
a, WB analysis of the subcellular localization of endogenous MBD2c in MCF-10A, HBL-100, PLC, Huh7, THLE-3 and HEK-293T cells. Protein levels were assessed in whole-cell lysate (WCL), mitochondrial (Mito.), cytosolic (Cyto.), and nuclear (Nucl.) fractions, verified by Lamin B, Tubulin, and TOM20 protein levels as WB loading controls. b, WB analysis of the subcellular localization of Flag-tagged MBD2c in MDA-MB-468 and MDA-MB-231 cells. c, Schematic of MBD2c protein structure and selected deletion variants. Subcellular localization of Flag-MBD2c and indicated deletion variants in MDA-MB-468 cells was assessed by WB using anti-Flag antibodies in WCL and purified mitochondrial fraction, validated by Tubulin, Lamin B, and TOM20 protein levels. d, WB analysis of subcellular localization of endogenous MBD2c in MDA-MB-468 cells stably expressing NTC or shTOM20. VDAC was used as the loading control. e, HEK-293T cells were co-transfected with HA-TOM20 and Flag-MBD2c-WT or Flag-MBD2c-ΔC plasmids. Co-IP experiment was conducted using IgG or anti-Flag antibodies, followed by WB analysis. f, ChIP experiments were performed in MDA-MB-468 cells stably overexpressing Flag-MBD2c using IgG or anti-Flag antibodies. The noncoding region (NCR) occupancy by MBD2c was determined by qPCR, with atp8 and mt-co1 as the negative controls (n = 3 samples). g, ChIP-seq experiments conducted in MDA-MB-468 cells stably overexpressing Flag-tagged MTS-MBD2c, further infected with shTFAM-expressing lentivirus by using IgG or anti-Flag antibodies. Integrative Genomics Viewer (IGV) representation of ChIP-seq of MBD2c or BS-seq of 5mC displaying distribution encompassing the entire mitochondrial genome. The red line indicates the sequence of 5mC-modified HSP probes used in Fig. 1i. h, MedIP or ChIP&MedIP assays were conducted in MDA-MB-468 cells stably overexpressing Flag-tagged MTS-MBD2c. MedIP was carried out with IgG or anti-5mC antibodies. 5mC occupancy on NCR was assessed by qPCR (n = 3 samples). Immunoblots are representative of three independent experiments (a-e). In immunoprecipitations, approximately 10% of the supernatant was collected as input and equal volume of input and elution were loaded for WB. Error bars denote the mean ± S.E.M. (f,h). Statistical analyses were performed by two-tailed Student’s t-test (f,h).
Extended Data Fig. 2 MBD2c interacts with TFAM to promote mitochondrial transcription and respiration.
a-b, mtDNA-encoded mRNA levels and mtDNA contents were determined by qPCR in MDA-MB-468 cells with MBD2c KD (n = 3 samples). c, WB analysis of TFAM, TFB2M and POLRMT in MDA-MB-468 cells expressing NTC or shMBD2c. Actin served as the loading control. d, WB analysis of mtDNA-encoded protein levels in MDA-MB-231 and T-47D cells stably expressing NTC or shMBD2c. Actin served as the loading control. e, MDA-MB-468 cells with stable knockout of endogenous MBD2c, were further infected with viruses expressing EV, Flag-tagged MBD2c-WT, MTS-MBD2c or MBD2c-ΔC. WB analysis of subcellular localization of endogenous MBD2c and mtDNA-encoded proteins expression. Actin and TOM20 served as loading controls of whole-cell lysates or mitochondrial proteins, respectively. f, MDA-MB-468 cells expressing NTC or shMBD2c were further infected EV or Flag-tagged MTS-MBD2c lentivirus, followed by WB analysis to detect mtDNA-encoded proteins. Actin served as the loading control. g, WB analysis of mtDNA-encoded proteins levels in MDA-MB-468 cells stably expressing NTC or shTOM20. Actin served as the loading control. h, Co-IP assay was performed in HEK-293T cells transfected with Flag-TFAM and HA-MBD2c plasmids using anti-Flag antibodies. i, ChIP experiments were performed in MDA-MB-231 cells stably overexpressing Flag-TFAM, further infected with NTC or shMBD2c lentivirus by using IgG or anti-Flag antibodies. The NCR occupancy by TFAM was determined by qPCR (n = 3 samples). j, WB analysis of MBD2c and TFAM protein levels in MDA-MB-468 or MDA-MB-231 cells overexpressing Flag-TFAM that further infected with NTC or shMBD2c lentivirus. k, WB analysis of mtDNA-encoded protein levels in MDA-MB-468 cells stably expressing NTC or shTFAM. Actin served as the loading control. l-m, Cellular ATP concentrations and mitochondrial membrane potential (MMP) were detected in MDA-MB-468-shMBD2c cells further infected with EV or Flag-TFAM lentivirus (n = 3 samples). Immunoblots are representative of three independent experiments (c-h,j,k). In immunoprecipitations, approximately 10% of the supernatant was collected as input and equal volume of input and elution were loaded for WB. Error bars denote the mean ± S.E.M. (a,b,i,l,m). Statistical analyses were performed by two-tailed Student’s t-test (a,b,i,l,m).
Extended Data Fig. 3 MBD2c recruits SIRT3 to deacetylate and activate TFAM.
a, IP assay was performed using anti-TFAM antibodies in isolated mitochondria fractions from MDA-MB-468 cells treated with 10 mM NAM for 24 h, followed by WB analysis. b, IP assay using anti-MBD2c antibodies, was performed in MDA-MB-468 cells pre-treated by DSP, with or without KCC-07, followed by WB analysis. c, IP assay was performed in isolated mitochondria fractions using anti-MBD2c antibodies, with or without DNase I, followed by WB analysis. d, WB analysis of the levels of mtDNA-encoded proteins in MDA-MB-468 cells stably expressing NTC or shSIRT3. e, ChIP-qPCR was performed in MDA-MB-468 Flag-TFAM cells treated with KCC-07 or 3-TYP by using IgG or anti-Flag antibodies. The NCR occupancy by TFAM was determined by qPCR (n = 3 samples). f, IP was performed with anti-IgG or anti-Flag antibodies in cells mentioned in (e), followed by WB analysis. g, The diagram illustrates potential acetylated sites and structural ___domain of TFAM (upper panel). ChIP experiments were performed in MDA-MB-468 cells overexpressing Flag-tagged TFAMWT, TFAMK51&95&111Q, TFAMK145&146&154Q, and TFAMK181Q by using IgG or anti-Flag antibodies. The NCR occupancy was determined by qPCR (lower panel, n = 3 samples). h, MDA-MB-468-shMBD2c cells with stable expressing Flag-tagged TFAMWT, TFAMK145Q and TFAMK146Q, were further infected with sgTFAM viruses followed by WB analysis of mtDNA-encoded protein levels. i, qPCR detection to determine the mtDNA content in the cells mentioned in (h) (n = 3 samples). j, MDA-MB-468 cells with stably expressing EV, Flag-tagged TFAMWT, TFAMDKR and TFAMDKQ, were further infected with viruses to knockout endogenous TFAM, followed by qPCR detection of mt-RNA levels (n = 3 samples). k, MDA-MB-468 cells stably overexpressing Flag-tagged TFAMWT, or TFAMDKQ were further infected with NTC, shMBD2 or shSIRT3 lentivirus. IP was conducted using anti-Flag antibodies, followed by WB analysis. l, OCR were detected in cells mentioned in (k). The OCR curve was analyzed for basal respiration, maximal respiration, and ATP-linked respiration (n = 3 samples). Immunoblots are representative of three independent experiments (a-d,f,h,j). Error bars denote the mean ± S.E.M. (e,g,i,j,l). Statistical analyses were performed by two-tailed Student’s t-test (e,g,i,j,l).
Extended Data Fig. 4 MBD2c-enhanced mitochondrial activity contributes to drug resistance in TNBC.
a, WB analysis of the proteins encoded by the nuclear genome in MDA-MB-468 cells treated with 4 μM or 10 μM CDDP for 72 or 96 h. b, Cell growth curves were analyzed by trypan blue counting in MDA-MB-468 cells treated with 4 μM CDDP and/or 5 μM GSH (n = 3 samples). c, Detection of OCR in MDA-MB-468 cells treated with 4 μM CDDP and/or 5 μM GSH for 72 h (n = 3 samples). d, Measurement of mitochondrial ROS levels using MitoSOX staining and flow cytometry in MDA-MB-468 cells mentioned in (b) (n = 3 samples). e, Calculation of IC50 curves for CDDP in MDA-MB-468 cells stably expressing EV or MTS-MBD2c (n = 3 samples). f, qPCR analysis of mtRNA levels in MDA-MB-468 cells expressing EV or MTS-MBD2c treated with 4 μM CDDP for 72 h (n = 3 samples). Immunoblots are representative of three independent experiments (a). Error bars denote the mean ± S.E.M. (b,c,e,f). Statistical analyses were performed by two-tailed Student’s t-test (f) or two-way ANOVA with Tukey’s multiple comparisons test (b).
Extended Data Fig. 5 TFAM acetylation enhanced breast cancer cell sensitivity to CDDP.
a, Measurement of cell death ratio in MDA-MB-468 cells expressing NTC or shMBD2c in the absence or presence of 4 μM CDDP (n = 3 samples). b, Plate colony formation assays were performed to measure the effect of CDDP (4 μM) on cell growth in the MDA-MB-468 cells expressing NTC or shMBD2c. c, Subcutaneous injection of MDA-MB-468 cells into nude mice. Intraperitoneal administration of 4 mg/kg CDDP began from day 10, and tumor growth curves were measured simultaneously (n = 5 mice in each group). d, WB analysis of mtDNA-encoded proteins in tumor tissues treated with or without 4 mg/kg CDDP. Actin served as the loading control. e, Tumor weights were calculated at the end of the experiment as shown in (Fig. 5g). f, WB analysis of TFAM, MBD2c, and mtDNA-encoded proteins in tumor tissues from each group as illustrated in (Fig. 5g). Actin served as the loading control. Plate colony formation assays (b) and immunoblots (d,f) are representative of three independent experiments. Error bars denote the mean ± S.E.M. (a,c,e). Statistical analyses were performed by two-tailed Student’s t-test (a,e) or two-way ANOVA with Tukey’s multiple comparisons test (c).
Extended Data Fig. 6 Gating strategies used in cell death analysis.
The gating strategy to identify the proportion of the cell death.
Supplementary information
Supplementary Information
Supplementary Tables 1–8.
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Hao, Y., Zhou, Z., Liu, R. et al. Mitochondria-localized MBD2c facilitates mtDNA transcription and drug resistance. Nat Chem Biol 21, 926–938 (2025). https://doi.org/10.1038/s41589-024-01776-1
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DOI: https://doi.org/10.1038/s41589-024-01776-1
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