Role of post-translational modification in KRAS expression: interplay between PARP1, Reactive Oxygen Species and G-quadruplex DNA

Over 90% pancreatic ductal adenocarcinomas (PDACs) carry mutations in exon 1 of the KRAS gene causing the malignant transformation of the cells. One main function of mutant KRAS is to reprogram the metabolism in PDAC to generate biomass and reducing power to fuel a high proliferation rate. Previous studies have demonstrated that the KRAS promoter contains a G-rich sequence located immediately upstream of the transcription start site (TSS). Our laboratory reported for the first time in 2006 that this sequence (called 32R or G4 near) can fold in a non-canonical DNA structure, called G-quadruplex (or G4) under physiological conditions. Two years later, we found that the KRAS G4 is recognized by several transcription factors (TFs) including MAZ, hnRNP A1, Ku70 and PARP1. PDAC cells produce more reactive oxygen species (ROS) than healthy cells, leading to oxidation of lipids, proteins and DNA. Among the four nucleobases, guanine is more prone to oxidation to 7,8-dihydro-8-oxoguanine (8OG). Previous data from our laboratory revealed that an increase of oxidative stress results in a higher level of 8OG in the promoter G4 region than in non-G4 guanine-rich regions. This guanine lesion is likely to behave as an epigenetic mark for the recruitment of MAZ and hnRNP A1 to the promoter. A crucial report in this thesis is the existence of a strict correlation between KRAS and oxidative stress, as an increase of ROS significantly stimulates the expression of the gene. We reasoned that PARP1 could control transcription because when ROS are enhanced in PDAC cells, PARP1 is recruited to the KRAS promoter in the region near to TSS. PARP1 (Poly-ADP-Ribose Polymerase 1) is a nuclear protein that catalyses the transfer of poly ADP-ribose units (PARylation) or mono ADP-ribose unit (MARylation) onto target proteins, including itself. PARP1 is known to take part to the base excision repair (BER) mechanism by which an oxidized base is recognized, excised and repaired. We found that PARP1 tightly binds to 32R. Mobility-shift assays (EMSA) ...