Demethylation of H3K4me3 is a necessary step to allow DNA double strand breaks repair.

BAYO J; MALVICINI M; RIZZO M

Congreso; Reunión Anual de Sociedades de Biociencia (SAIC SAFE SAB SAP); 2019

Background: Human genome is highly susceptible to DNA damaging agents. Double-strand breaks (DSBs) are considered the most cytotoxic form of DNA damage. As a consequence to DSB, DNA damage response (DDR) initiates repair mechanisms including homologous recombination (HR) and non-homologous end joined (NHEJ). Epigenetic involves mechanisms that affect DNA-based processes such as DSB repair through changes in the chromatin. Particularly, Jumonji (JmjC) histone lysine demethylases (KDM) play roles in DNA repair pathways. Our aim is to study if demethylation of histones is a required step during the DSB repair. Methods: The efficiency of repair by NHEJ and HR were assayed with a plasmid-based reporter system. DSB were induced by using ionizing radiation on H1299 cancer cell line. DNA repair was determined by immunofluorescence against gH2AX and 53BP1. Demethylation abrogation was performed using pharmacological inhibitors (JIB-04, PBIT, GSK-J4) or performing siRNA knockdown (KD) experiments. Rescue experiments were performed by electroporating H1299 cells with a plasmid codifying for JARID1B. Demethylases activity was assayed using an Epigentek commercial kits. CHIP-qPCR experiment was performed using a Millipore commercial Kit. Histone methylation levels were assayed by western blot (WB). Results: The efficiency of repair by NHEJ and HR is inhibited by JIB-04, a JmjC pan-inhibitor. In addition, treatment with JIB-04 and inhibitors of H3K4me3 KDM (PBIT) but not of H3K27me3 KDM (GSK-J4) delay the resolution of DSB marked by gH2AX and 53BP1 foci (p