Project description:To identify mutations that occurred in the nuclear and mitochondrial DNA of the yeast subjected to mtDNA base editing or Mito-BE screen, we performed whole-genome sequencing of cultured yeast cells after isolation of mitochondrial DNA.
Project description:To study whether increase in mitochondrial oxidative stress (SOD2 removal) and decrease in mitochondrial DNA repair (Ogg1 dMTS) results into increase in mitochondrial DNA mutation load. Oxidative stress has been suggested to induce mutations in mtDNA. To verify this, we extracted and sequenced (Illumina) mitochondrial DNA from heart Sod2 knockout animals that were also deficient for mitochondrial base-excision repair. The repair deficiency was induced by removing the genomic region encoding for the predicted mitochondrial targeting sequence from endogenous OGG1 (L2 to W23) called Ogg1 dMTS mice, thus excluding the protein from mitochondria. OGG1 is a DNA glycosylase that recognizes and repairs 8-oxo-dG damage from DNA. Oxidative stress can induce 8-oxo-dG lesions, thus we removed the mitochondrial matrix localized superoxide dismutase (SOD2) from these mice to increase the level of oxidative stress. 8-oxo-dG lesion can be mutagenic because some DNA repair polymerases are known to erroneously incorporate adenosine opposite to 8-oxo-dG during replication leading to GC>TA transversion mutations.
Project description:Studying whether removal of base-excision repair from mitochondria will result into increase in mitochondrial DNA (mtDNA) mutation load. The endogenous genes of OGG1 and MUTYH DNA glycosylases were modified to lack the genomic region encoding for the predicted mitochondrial targeting sequence. The mouse lines used: A mouse line that lacks the region encoding for the mitochondrial targeting sequence (L2 to W23) of OGG1 (Ogg1 dMTS mice). A mouse line that lacks the region encoding the mitochondrial targeting sequence (K2 to P33) of MUTYH (Mutyh dMTS mice). To accumulate mutations to the mitochondrial DNA these mice were bred double homozygous Mutyh dMTS x Ogg1 dMTS mice as a maternal lineage for five consecutive generations and mitochondrial DNA from liver was extracted from the offspring and sequenced with Illumina. OGG1 and MUTYH are involved in repair of 8-oxo-dG from DNA. 8-oxo-dG can be a mutagenic lesion because some DNA repair polymerases are known to erroneously incorporate adenosine opposite to 8-oxo-dG during replication leading to GC>TA transversion mutations.
Project description: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.
Project description:The transcription cofactor Yki drives growth and proliferation in part by controlling mitochondrial network formation. To determine if Yki and Sd are directly bound to DNA corresponding to mitochondrial genes, we used chromatin immunoprecipitation and whole genome tiling arrays (ChIP-chip) to identify regions bound by these factors in eye-antenna and wing imaginal discs. The supplementary .bed files contain all Yki or Sd binding sites (called at 5% FDR) in wing or eye-antenna imaginal discs, as well as shared Sd+Yki sites and associated target genes.