Breaking silence: Investigating DNA double-strand break repair in heterochromatin

Our genetic material is constantly exposed to damaging influences from both external and internal factors such as UV radiation and metabolic byproducts. This thesis focuses on DNA double-strand breaks (DSBs), a severe form of damage in which both strands of the DNA helix are broken. If not repaired accurately or in a timely manner, DSBs can accumulate and lead to genomic instability due to incorrect repair. In eukaryotic cells, DSBs are mainly repaired through two pathways: non-homologous end joining (NHEJ), which directly ligates broken DNA ends, and homologous recombination (HR), which uses an intact homologous DNA sequence as a template for precise repair.
Beyond the DNA itself, damage also affects the surrounding chromatin environment, where DNA is packaged with histone proteins. Chemical modifications of histones and their interactions with regulatory proteins determine chromatin structure and function. Chromatin exists in two major forms: euchromatin, which is relatively open and transcriptionally active, and heterochromatin, which is compact, transcriptionally inactive, and enriched in repetitive sequences. Although heterochromatin plays a limited role in gene expression, it is essential for maintaining genome stability and nuclear organization. However, DSBs occurring in heterochromatin are more prone to erroneous repair, partly due to the repetitive nature of its DNA. These breaks are often relocated to the periphery of heterochromatic domains before repair, highlighting fundamental differences in repair mechanisms between chromatin types.
This thesis investigates how heterochromatin responds to DSBs and how these responses influence repair pathway choice and break relocation. It shows that, following DNA damage, heterochromatin undergoes dynamic changes, including increased acetylation of histone H3 at lysine 9 (H3K9ac) mediated by dGcn5. This modification is important for efficient repair, as its absence delays the relocation of breaks and impairs recruitment of key repair factors.
Using a newly developed in vitro system with synthetic chromatin structures that mimic euchromatin and heterochromatin, this work demonstrates that the pre-existing chromatin state directly influences the binding of repair proteins. While the NHEJ factor 53BP1 preferentially associates with damaged euchromatin, several HR-related proteins show a preference for damaged heterochromatin. In addition, heterochromatin temporarily loses its characteristic proteins at sites of damage, suggesting a transition toward a more open, euchromatin-like state to facilitate repair.
Finally, functional analysis of candidate proteins identified in this system reveals that ZNF445 plays an important role in maintaining heterochromatin integrity. Loss of this protein leads to structural changes in heterochromatin and accumulation of DNA damage, underscoring its role in genome stability.
Overall, this work highlights the dynamic nature of heterochromatin during DNA damage and repair, and demonstrates how chromatin context influences both the mechanism and efficiency of DSB repair.