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. 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.