Fierz, BeatFranz, Pauline2024-07-022024-07-02202410.5075/epfl-thesis-10618https://infoscience.epfl.ch/handle/20.500.14299/208942DNA damage signaling following DNA double-strand breaks (DSBs) involves numerous regulating proteins, which dynamically recognize ('read') and alter ('write' or 'erase') histone post-translational modifications (PTMs). Among these PTMs, the ubiquitin system plays a key role in the two major pathways of DSB repair, homologous recombination (HR) and non-homologous end-joining (NHEJ), which are deregulated in many diseases, especially in cancer. Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DSB repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, mechanistic insights into how ubiquitin recognition by RNF168 affects H2AK15Ub deposition have remained elusive. This work aimed to examine the role of the E3 ubiquitin ligase RNF168 as a reader and writer of ubiquitylation marks in DNA repair from a chemical biology perspective. Here, I developed a method to chemically site-specifically di-ubiquitylate H1 on four different DNA repair-associated ubiquitylation hotspots on H1 (H1KxUb2, x = 17, 46, 64, 97). Incorporated H1KxUb2 variants in 'designer' chromatin fibers via in vitro reconstitution revealed the simulation of the E3 ligase activity of RNF168. Strikingly, the presence of H1KxUb2 enhanced the ubiquitylation activity of RNF168 compared to H1-containing and unmodified chromatin arrays, suggesting an effect beyond chromatin fiber opening. The stimulation of RNF168's ubiquitylation activity depended on its ubiquitin-binding capacity, confirming that ubiquitin recognition of di-ubiquitin on H1 was the driving force underlying the observed increase in ubiquitylation activity. Furthermore, the stimulatory effect of RNF168 ubiquitylation activity depended on the attachment site of ubiquitin moieties on H1, in particular, position 17 (H1K17Ub2). Finally, I studied the behavior of RNF168 in the presence of H1K17Ub2 in cellulo to gain insights into its binding behavior in a more complex environment. As the cellular context precludes selective control over PTMs, the previously synthesized, non-hydrolyzable triazole-linked H1K17Ub2 was introduced via bead-loading into U-2 OS cells, which displayed a dispersed nuclear distribution. With increasing bead-loading efficiencies of H1K17Ub2 (but not H1), RNF168 nuclear foci formation was disrupted, even in the presence of DNA-damaging treatment. Thus, H1K17Ub2 likely delocalized RNF168, thereby impeding its interaction with endogenous ubiquitin marks, such as poly-ubiquitylated H1, necessary for proper foci recruitment. Together, these observations indicate that K63-linked ubiquitin installed on H1 directs RNF168 recruitment to chromatin in cells. Overall, this work provides mechanistic insights into the crosstalk of H1 and H2A ubiquitylation via RNF168. In particular, the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers. In summary, the results strengthen the hypothesis that the direct binding of poly-ubiquitylated H1 by the RNF168 UDM1 domain is a key driving force underlying RNF168 recruitment to DNA damage sites.enchromatinubiquitylationchemical biologybiochemistrymolecular mechanismspost-translational modificationschemical ubiquitylationDNA repairthesis::doctoral thesis