Ubiquitinated H2A.X-Induced RNF168 Condensation Promotes DNA Double-Strand Break Repair and Tumor Radioresistance

Abstract

Ubiquitination of histone is an essential process involved in DNA damage response (DSB) serving as scaffolds for DNA repair proteins, but how these factors are recruited so quickly and regulated in a spatiotemporal manner remains poorly understood. Liquid-liquid phase separation (LLPS) has recently emerged as a mechanism for membraneless condensation driven by multivalent interactions. In this study, we aimed to investigate the LLPS potential of RNF168, an E3 ligase essential for DSB repair, and the mechanism underlying its-mediated tumor radio-resistance. The intrinsic disordered domain (IDR) of RNF168 was determined by the PONDR website. The LLPS properties were validated by droplet formation in vivo and in vitro. RNF168-mEGFP were expressed in Escherichia coli and purified with GST tag. The synthesized K63-linked ubiquitin chains were added to mimic the interactions between RNF168 and radiation-induced ubiquitinated-histone. Effects of RNF168 LLPS on downstream proteins were verified by immunofluorescence. RNF168-mEGFP recombinant protein formed liquid-like droplets in vivo and co-localized with γ-H2A.X foci after irradiation. The droplet's fluorescence recovered quickly after photobleaching, which could be abolished by 1,6-hexanediol treatment or ATP deprivation. Purified RNF168-mEGFP protein also condensed in vitro, and the size and number of droplets were related to protein concentration, salt concentration, pH, and temperature. Condensation of RNF168 was dependent on the IDR (323-459 amino acid), and more importantly, enhanced by synthesized K63-linked ubiquitin chains. LLPS of RNF168 was required for recruitment of RNF168 to DSB and RNF168-mediated γ-H2A.X ubiquitination. LLPS deficiency of RNF168 resulted in decreased recruitment of 53BP1, BRCA1, and RAP80 proteins, resulting in impaired DSB repair and genomic instability. Notably, higher expression of RNF168 was correlated with a poorer response to neoadjuvant radiochemotherapy in rectal cancer patients. Finally, RNF168 condensate-induced tumor radioresistance was further verified in the xenograft model. RNF168 undergoes LLPS at the DSB site, which is determined by both the IDR domain and the interaction with K63-linked ubiquitin chains. Radiation-induced RNF168 condensation accelerates the accumulation of RNF168 and promotes the recruitment of downstream effectors to DSB, resulting in enhanced DSB repair and tumor radioresistance.