ChIP-Seq Worm_Lonp1_IP_vs_IgG

We performed the CHIP-SEQ assay for LONP1 in C.elegans. Our findings suggest an evolutionary conserved mechanism where mtDNA-bound LONP1 serves as an internal sensor of organelle function that promotes mtDNA replication in dysfunctional compartments. We propose that if LONP1 activity declines, ATFS-1 avoids degradation and binds mtDNA to promote replication in an effort to recover the dysfunctional compartment, which inadvertently promotes ?mtDNA replication in heteroplasmic cells. Overall design: Synchronized worms were cultured in liquid and harvested at early L4 stage by sucrose flotation. The worms were lysed via Teflon homogenizer in cold PBS with protease inhibitors (Roche). Cross-linking of DNA and protein was performed by treating the worms with 1.85% formaldehyde for 15 min. Glycine was added to a final concentration of 125mM for 5 min at room temperature to quench the formaldehyde. The pellets were resuspended twice in cold PBS with protease inhibitor. Samples were sonicated in Bioruptor (Diagenode) for 15 min at 4°C on high intensity (30s on and 30s off). Samples were transferred to microfuge tubes and spun at 15,000*g for 15 min at 4°C. The supernatant was precleaned with pre-blocked ChIP-grade Pierce™ magnetic protein A/G (Thermo Scientific) and then incubated with Monoclonal ANTI-FLAG® M2 antibody (Sigma, F1804) or Mouse mAb IgG1 Isotype Control (Cell Signaling Technology, G3A1) rotating overnight at 4°C. Then, the antibody-chromatin complex was precipitated with magnetic beads. After washing, the crosslinks were reversed by incubation at 65 °C overnight and further treated with RNaseA 37 °C for 1 hour and then proteinase K 55 °C for 2 hours, respectively. Finally, immunoprecipitated and input DNA were purified with ChIP DNA Clean & Concentrator (Zymo Research, D5205) and used as templates for qPCR or next generation sequencing.