DNA double strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The choice of the DSB repair pathway depends on which proteins bind DSB ends and how their activities are regulated. NHEJ requires the Ku complex binding to the ends, while HR initiates with the nucleolytic degradation of the 5’-endeded strands (resection) by several DNA nucleases/helicases. Resection generates 3’-ended single-stranded DNA tails that commit repair by HR, while destroying the NHEJ substrate. DSB repair occurs within a precisely organized chromatin environment, where DNA is wrapped around histones to form nucleosomes. Nucleosomes impose a barrier to the DNA repair machinery and particularly to the resection. Chromatin organization around a DSB is modified to allow proper DSB repair by either the removal of entire nucleosomes thank to the action of chromatin remodeling factors and/or by post-translational modifications (PTMs) of histones, thus increasing chromatin flexibility and accessibility to DNA of repair enzymes. Several histone PTMs have been characterized for their role in promoting different repair pathways, however a comprehensive view of the complex interplays among histone PTMs and DSB repair factors is still elusive. The overall goal of this project is to use a combination of genetic and biochemical approaches in the model eukaryote Saccharomyces cerevisiae to expand our knowledge on the involvement of histone PTMs, particularly H3/H4 lysine methylation and acetylation, in DSB response with particular attention to their role in DSB repair pathway choice. The contribution of histone H3/H4 PTMs in DSB response will be explored by either searching for genetic relationships among mutations affecting DSB repair pathways and a panel of H3 and H4 histone mutations (Task 1.1) and defining the landscape of H3/H4 PTMs in the surroundings of a DSB (Task 1.2). In addition, the contribution of histone PTMs in regulating the DNA association of the Rad9 regulator will be tested (Task 2.1), as well as the effects on DSB response caused by the inactivation of H3/H4 histone methylases, demethylases, acetylases, and deacetylases obtained by either genetic manipulation (Task 2.2) or pharmacological treatments (Task 2.3). This experimental plan will be feasible thanks to the close collaboration between two research units, which have complementary expertise and already set up most of the yeast-based systems and technical field required to achieve this proposal. The knowledge acquired with this fundamental research plan could support or revise the hypothetical models recently proposed for the mammalian system and provide high-throughput methodologies for initial validation of single and combination epigenetic therapies based on PTMs inhibition.