Fig. 5: NAD+-induced PARylation stimulates mtDNA transcription.

a TFAM-ChIP and ADPr-ChAP (PAR) were performed on mitochondria, and the sequencing reads were mapped onto the mitochondrial genome. Key mtDNA genes are shown. b ADPr-ChAP and TFAM-ChIP seq signal intensities of 37 mitochondrial genes at their respective gene start sites were calculated by seqMINER (Ver 1.3.3) and plotted. c HeLa WT and PARP1-KO cells were subjected to TFAM-ChIP. Two-way ANOVA was used to calculate the p value. d U2OS WT/PARP1-knockdown (KD) cells were treated with DMSO, 1 mM NR and 1 mM NR plus 5 µM olaparib for 24 h, followed by immunoprecipitation with normal rabbit IgG or TFAM antibodies. The immunoprecipitated products analyzed by western blotting with the indicated antibodies. e, f Isolated mitochondria from WT and PARP1-KO cells were treated with NAD + with/without olaparib for 30 min at 37 °C. Subsequently, mitochondrial samples were treated with RNase A to eliminate residual nuclear/cytoplasmic RNA. After the treatment, mitochondrial RNA was purified and reverse transcribed (n = 5). g U2OS cells were treated with NR with/without olaparib for 24 h. TFAM-ChIP was performed with purified mitochondria (n = 3). qPCR was performed with primers specific to the mtDNA D-loop locus. Error bars represent the standard deviation. *, <0.05; **, <0.01; ***, <0.001. h Schematic diagram of NAD⁺-induced PARylation stimulating mtDNA transcription. See text for details.