XICUT · Epigenome establishment and maintenance of the inactive X chromosome in response to DNA double-strand breaks in mammalian cells

Epigenome establishment and maintenance of the inactive X chromosome in response to DNA double-strand breaks in mammalian cells

The DNA damage response takes place on a chromatin substrate that needs to be disorganized to allow access to the repair factors to the DNA lesion, then restored to preserve epigenome integrity. The chromatin structure differs according to the genomic region, with features impacting DNA repair, both at the molecular level and at the level of the repair pathway choice, and chromatin dynamics that accompany the DNA damage response. In mammalian female cells, one of the two X chromosomes is inactivated early on during embryonic development by the formation of facultative heterochromatin, which molecular components such as post-translational histone modifications and associated proteins are well characterized. My research project aims at identifying changes in the chromatin organization that accompany the repair of DNA double-strand breaks, and the mechanisms at play in maintaining the structure and function of the chromatin associated to the inactive X chromosome during DNA double-strand break repair. For that, I induce DNA double-strand breaks on the X chromosome of human female cells using the CRISPR/Cas9 technology, that allows sequence-specific targeting of the Cas9 nuclease. After repair is complete, I analyze the mutational signatures on the targeted genomic regions, because these genomic instability profiles are a proxy of the DNA repair pathway at play. I study the dynamics of the chromatin marks associated with the inactive X chromosome using immunofluorescence and chromatin immunoprecipitation approaches, to better understand the interplay between facultative heterochromatin dynamics and DNA double-strand break repair pathway choice. Moreover, the inactivation of one of the two X chromosomes, which is a crucial process for cell homeostasis, is random and occurs in embryonic stem cells that proliferate rapidly and are therefore prone to replication stress. This replication stress induced DNA double-strand breaks that could play a role in the choice of the X chromosome that will be inactivated, and could then impact stem cell differentiation. As such, I study the impact of DNA double-strand breaks occurring in the X chromosome of stem cells on their differentiation potential and their propensity to inactivate one of the two X chromosomes.

Data: CORDIS, © European Union