Mechanistic principles of permanent epigenetic silencing and their exploitation to improve targeted epigenetic editing

The molecular mechanisms that regulate gene expression within our cells represent a valid tool to inactivate therapeutically genes that, when mutated, induce diseases in humans. Starting from these mechanisms, we have recently developed a platform for targeted gene silencing based on epigenetics, one of the processes that controls gene expression. This platform exploits Engineered Transcriptional Repressors (ETRs), engineered proteins composed of a programmable DNA binding domain and epigenetic repressors. Once delivered into the cell, the ETRs recognize the gene for which they have been programmed to and recruit a multitude of cellular proteins that, in collaboration with the ETRs, switch off their target gene (Amabile et al., Cell. 2016). Given the dependence of the ETR technology on the molecular machinery of the cell, understanding these processes could provide relevant information for developing more effective and safe targeted gene silencing platforms. The goal of this project is to identify the factors that regulate ETRs’ activity, initially using large-scale genetic loss-of-function screening and cell lines that faithfully report for the gene silencing processes induced by ETRs. Based on these results, we will then characterize the molecular function of the identified factors in experimental models relevant to these processes. At the same time, we will evaluate whether inactivation or expression of the factors identified above in cells that are poorly responsive to ETRs increases their function. Beyond their biological relevance, these studies will allow developing improved versions of our silencing platform, prompting its application in human cells of therapeutic relevance, a fundamental step for the clinical translation of targeted epigenetic silencing.