Periodic Reporting for period 2 - EpigeneticScars (Understanding DSB repair from pathway choice to long term effects and their consequences.)
Berichtszeitraum: 2022-02-01 bis 2023-07-31
Cells have two main DSB-repair pathways, an error-free repair known as homology recombination (HR) and non-homologous end joining (NHEJ), which usually generates mutations. Both mechanisms are selectively used, and the activity of the sensor proteins arriving at the DSB site has a major role in the repair outcome.
We investigated the sensor-dependent interaction networks of three sensors in a time-dependent manner (Sirt6, Ku80, and NBS1). This could lead to important proteins to resolve DNA damage and prevent age-related diseases Using a proximity-labeling assay (BioID) and IP-mass spec, we identified chromatin networks for each sensor during different DDR time points. Our results showed that after DSB induction, the basal interactome of each sensor protein changes, progressively incorporating new proteins and approaching a similar state that requires functions like Nucleolar metabolism, RNA processing and degradation, and chromatin remodeling. Each sensor has its independent interactome and the shared interactome of DDR repair proteins. These interactome network will help us understand the DDR in a sensor dependent manner, to identify new relevant pathways and proteins involved that could be relevant for age-related diseases. In the following months we will focus on these novel proteins and pathways to understand their roles in DDR.
Regarding the Epigenetic signature, the material obtained in mass spectrometry was insufficient. Therefore, we are changing our protocol from acid to High salt extraction to obtain better histone purification. Last, we performed ChIP analysis on SIRT6 and Ku80 cell lines with and without damage. The data is being analyzed, but it is possible that this method will not be sensitive enough from our preliminary analysis, and we would need to implement an improved sequencing method.