The spatial regulation of genetic and epigenetic integrity in Embryonic Stem cells

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DNA is unstable and it can bear different types of lesions. We study breaks which affect both strands of DNA, the Double Strand Breaks (DSBs). DSBs can be repaired in an error free manner by copying the information from the sister chromatin using Homologous recombination. But they can also lead to deletions which can be either small if Non Homologous End Joining (NHEJ) is used, or larger if the ends are re-joined using the microhomology found in the two sides of the break, via Microhomology Mediated End Joining (MMEJ). Deletions signatures from these pathways have been found in cancer genomes. DNA is in the form of chromatin and chromatin is not linear, but it is folded in three dimensions and associates with different nuclear compartments which further specify chromatin characteristics. A DSB also leads to extensive remodelling of the chromatin structure around the break with histone exchange and alterations of histone modifications. Currently, it is unknown to what extend the epigenome is restored after DNA damage. Permanent genetic and epigenetic scars can alter gene expression profiles and are hallmark of cancer. If these scars occur in embryos or in stem cells, they can alter cell identity and the reprogramming potential. Our work together with work from other labs demonstrated that active chromatin is more prone to error-free repair and that compacted chromatin is more to MMEJ/NHEJ. Chromatin and 3D genome organization is cell type specific and dramatically changes during differentiation. In addition, stem cells bear a very unique chromatin feature which in called bivalency in which active and inactive chromatin marks exist together at the same nucleosome and decorates developmentally regulated promoters and protects them from DNA methylation observed in cancers.It is currently unknown:1. Do DNA repair pathways adapt to changes in chromatin 3D genome organization to confer a cell type specificity in DNA repair fidelity? 2. Is the epigenome fully restored after DNA damage?In this proposal we will use mouse Embryonic Stem cells and study the spatial regulation of DNA repair fidelity and how this changes upon differentiation in different lineages . Then we will investigate the mechanisms controlling DNA repair fidelity at each chromatin state. Finally, we will ask whether the changes at the chromatin structure are fully restored after DNA damage and study the impact of genetic and epigenetic scars in stem cell identity.Our proposal will elucidate the complex relationship between genome and epigenome integrity and its link to mutagenesis and cell identify. Recently, there has been considerable effort in developing genome editing methods which are based on generation of DNA lesions by CRISP Cas nucleases used in this proposal. Therefore, our results will be very valuable for medical and research purposes as detailed understanding of genome editing effectiveness and particularly fidelity and precision in embryonic stages and how this altered in adult tissues, is of paramount importance for correcting disease mutations at stem cells which can then differentiated in the lab to the tissue which is affected by a disease.