Mitochondria-localized MBD2c facilitates mtDNA transcription and drug resistance [Bisulfite-Seq]. Mitochondria-localized MBD2c facilitates mtDNA transcription and drug resistance [Bisulfite-Seq]

Mitochondria contain a 16kb-dsDNA genome encoding 13 proteins essential for respiration, whereas its regulatory mechanism and potential role in cancer development remain elusive. Although Methyl-CpG-binding protein (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. Specifically, MBD2c binds D-loop regions in mtDNA to recruit SIRT3, which in turn deacetylates TFAM, a primary mitochondrial transcription factor, and activates its function. TFAM activation subsequently enhances transcription of the whole mitochondrial genome. Furthermore, MBD2c overexpression recovered the decreased mtDNA-encoded RNA and protein levels induced by the DNA synthesis inhibitor, cisplatin (CDDP), in vitro and in vivo, preserving mitochondrial gene expression and respiration, consequently enhancing TNBC cells drug resistance and proliferation. 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. Overall design: Bisulfite-Seq (BS-seq) in purfied mtDNA from MDA-MB-468 cells, to facilitate site-specific identification of 5mC modifications in mitochondrial DNA.

线粒体拥有一段长16kb的双链DNA（double-stranded DNA, dsDNA）基因组，编码13种对呼吸作用至关重要的蛋白质，但其调控机制以及在癌症发生中的潜在作用仍不明确。
尽管甲基-CpG结合蛋白（Methyl-CpG-binding protein, MBD）对细胞核转录至关重要，但它们在线粒体DNA（mtDNA）转录中的功能仍未可知。
本研究报道，剪接变体MBD2c可易位进入线粒体，介导三阴性乳腺癌（TNBC）细胞中的mtDNA转录并增强线粒体呼吸作用。
具体而言，MBD2c结合mtDNA的D-loop区域，进而招募去乙酰化酶SIRT3；SIRT3可对核心线粒体转录因子TFAM进行去乙酰化修饰并激活其功能，TFAM的激活随后可增强整个线粒体基因组的转录。
此外，在体内外实验中，MBD2c过表达可恢复DNA合成抑制剂顺铂（cisplatin, CDDP）诱导下调的mtDNA编码RNA及蛋白质水平，维持线粒体基因表达与呼吸功能，最终增强三阴性乳腺癌细胞的耐药性与增殖能力。
上述研究结果共同证实，MBD2c可正向调控mtDNA转录，从而将去乙酰化介导的表观遗传调控与癌细胞代谢相联系，为克服癌症耐药性提供了可靶向干预的潜在治疗靶点。
整体实验设计：采用亚硫酸氢盐测序（Bisulfite-Seq, BS-seq）技术，对MDA-MB-468细胞纯化的mtDNA进行测序，以实现线粒体DNA中5-甲基胞嘧啶（5mC）修饰的位点特异性鉴定。