title: Novel regulators of chromatin response to DNA damage
Creators: name: Kolobynina, Ksenia G.
Publisher: name: UNSPECIFIED
Year: 2024

description: The DNA damage response (DDR) signaling maintains genome stability, thus protecting cells from aging or malignant transformation. DNA damage and following DDR occur in the context of chromatin, and chromatin governs the damage response at multiple levels including post-translational modifications of histone and non-histone proteins. Among these modifications reported at the sites of DNA double-strand breaks (DSBs) are phosphorylation, acetylation, methylation, and ubiquitination.
DSB signaling and repair take place in a defined chromatin domain characterized by the enrichment of specific post-translational modifications and accumulated proteins. These structures are microscopically visible and are termed “DNA repair foci". Phosphorylation of serine 139 of histone H2AX (termed γH2AX) is the key event and forms the platform for subsequent repair. The formation and persistence of γH2AX domains reflect chromatin architecture and the efficiency of DSB repair.
Another important post-translational modification involved in DSB repair is ubiquitination. Ubiquitination is a covalent protein modification, which requires a three-enzyme cascade. The high abundance of ubiquitin ligases, internal ubiquitin modification sites, and a variety of possible ubiquitin signal structures make ubiquitination one of the most complex modification types in DDR. Due to the complexity large part of the ubiquitin-dependent network remains to be discovered, although several ubiquitin ligases were shown to be involved in the DDR.
To identify novel ubiquitin ligases involved in the DDR a human ubiquitinome-wide (663 E3 ubiquitin ligases) high-throughput screening was performed using the formation and persistence of γH2AX foci as a proxy for DSB repair following X-ray exposure. More than 100 novel ubiquitin modifiers that affect DNA damage signaling (30 min post-irradiation) and/or repair (24 h post-irradiation) were identified in the screening. Of the identified hits 62.2% are associated with the damage signaling only (early time point, 107 ubiquitin modifiers), while 15.1 % of the hits are exclusively associated with the late repair point (24 h, 26 ubiquitin modifiers). The remaining 22.7% of the identified target genes affect both repair stages (39 ubiquitin modifiers). Gene ontology analysis and in silico protein-protein interaction analysis identified clusters of physically interacting hits for each time point. This data provided a list of novel chromatin modifiers involved in the DNA damage response and repair and their potential molecular function.
In a second screen 33 of the identified hits, which affected both early and late repair time points, were studied in more detail. This screen used more time points and additionally made use of the immunohistochemical detection of RAD51 (homologous recombination) and 53BP1 (non-homologous end joining) foci to gain information about the affected DSB repair pathways. In addition, we performed the cluster analysis and identified four time-independent DNA repair phenotypes represented by CXXC1, XIAP, RNF8 and RNF168 proteins. 
In the third part of this work, the molecular function of one hit, the PHF19, was studied in the context of the chromatin response to DSBs. PHF19 was shown to be crucial for the γH2AX signaling and formation of the repair focus. PHF19-depleted cells experienced global chromatin decondensation and a significant decrease in γH2AX foci intensity post-X-ray irradiation. Moreover, PHF19 knockdown caused DSB repair delay compared to wild type cells. In accordance with the γH2AX signaling impairment, the lack of the PHF19 protein hindered RAD51, RAD52, 53BP1, pATM and RNF8 recruitment to sites of DSBs. Both general ubiquitination and H2AK119ub specifically were decreased at the sites of breaks. With the use of ubiquitin binder probes, we showed that ubiquitination is reduced at the sites of the damage in living PHF19 knockdown cells. Additionally, we showed that PHF19 association with the DSB site reaches its maximum one hour post-irradiation.
In conclusion, these findings provide previously unknown ubiquitin-dependent signaling cascades in the DDR and underline the role of chromatin ubiquitination in DSB repair suggesting possible targets for anti-cancer therapy.