Specific inhibition of chromatin remodelers and its connection with defects in DNA repair after inducing DNA damage

Abstract

Genome integrity is continuously challenged by endogenous and exogenous agents causing DNA damage. To counteract the adverse consequences associated to DNA lesions, eukaryotic cells have developed DNA damage signaling and repair machineries specifically adapted to the type of damage. Defects in DNA damage response contribute to aging and several disorders, including cancer and neurodegenerative diseases, which highlights their critical importance for cell viability. Double-strand breaks (DSBs), the most deleterious form of DNA damage, occur in the context of a highly organized chromatin environment. The dynamics of DNA repair proteins, which in many cases form detectable foci, are dictated by chromatin organization and transcriptional activity. Because of that, all eukaryotic DNA repair pathways need to unravel the compacted chromatin structure to facilitate access of the repair machinery and restoration of the original chromatin state afterwards. However, how accessibility and transcriptional silencing are orchestrated and interpreted at DSB sites is still unclear. In order to study the connection between chromatin structure alterations and DSB repair foci formation, pharmacological inhibitors of histone deacetylases, acetylases, methylases or demethylases were used in tumor cell lines before and after inducing DNA damage by both ionizing radiation or Olaparib treatment, widely employed in cancer therapies. Their effect on the assembly of DSB repair foci were assessed later, focusing on sensor and mediator proteins, such as γH2AX, Nbs1, MDC1 or 53BP1. Our results seem to indicate that some HAT and KMT inhibitors impair the formation of 53BP1 foci, not the γH2AX ones, at damage sites. This supports the idea that specific histone acetylations and methylations are necessary for the assembly or stability of 53BP1 foci in resting cells. Further experiments are being performed to determine the molecular mechanism and the modifications of histones implicated in this process.