Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer

Abstract

Signaling and repair of DNA double-strand breaks (DSBs) are critical for preventing immunodeficiency and cancer. These DNA breaks result from exogenous and endogenous DNA insults but are also programmed to occur during physiological processes such as meiosis and immunoglobulin heavy chain (IgH) class switch recombination (CSR). Recent studies reported that the E3 ligase RNF8 plays important roles in propagating DNA DSB signals and thereby facilitating the recruitment of various DNA damage response proteins, such as 53BP1 and BRCA1, to sites of damage. Using mouse models for Rnf8 mutation, we report that Rnf8 deficiency leads to impaired spermatogenesis and increased sensitivity to ionizing radiation both in vitro and in vivo. We also demonstrate the existence of alternative Rnf8-independent mechanisms that respond to irradiation and accounts for the partial recruitment of 53bp1 to sites of DNA damage in activated Rnf8−/− B cells. Remarkably, IgH CSR is impaired in a gene dose-dependent manner in Rnf8 mutant mice, revealing that these mice are immunodeficient. In addition, Rnf8−/− mice exhibit increased genomic instability and elevated risks for tumorigenesis indicating that Rnf8 is a novel tumor suppressor. These data unravel the in vivo pleiotropic effects of Rnf8.

RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks

“…A recent study also suggested a role for RNF8 and RNF168 ubiquitylation and proteasomal degradation of JMJD2A in exposing H4K20me2 and facilitating 53BP1 recruitment to DSB sites (25). Although current data support the function of RNF168 in the DSB-signaling cascade downstream of RNF8, the requirement for this E3 ligase, but not RNF8 (3,8,11,12), for the initial recruitment of 53BP1 to DSB sites suggests that RNF168 also may function independently of RNF8 in the DSB-signaling cascade.…”

“…Furthermore, our data demonstrate the importance of 53BP1 ubiquitylation on K1268 for G2/M checkpoint activation, NHEJ repair of DSBs, and radiosensitivity. Consistent with RNF8-independent initial recruitment of 53BP1 to DSBs (8,11), RNF8 failed to interact with 53BP1 or mediate its ubiquitylation. In addition, no additive effect on 53BP1 ubiquitylation was observed in the presence of both RNF8 and RNF168.…”

“…With the Ubconjugating enzyme UBC13, RNF168 initiates ubiquitylation of lysine (K) 13 or 15 of histones H2A and H2A.X, leading to the recruitment of DDR proteins, including 53BP1, to DSBs. Although H2A.X, MDC1, and RNF8 are important for the retention of 53BP1 at these DSBs, its initial and transient recruitment to DNA breaks still occurs in their absence (8)(9)(10)(11). In contrast, deficiency of Rnf168 in mouse embryonic fibroblasts (MEFs) completely abolishes 53bp1 recruitment to DSB sites (12).…”

RNF4 is required for DNA double-strand break repair in vivo

“…We show that similar to the effect of null or hypomorphic mutations of genes involved in DSB signaling, such as H2Ax, 24 Mdc1, 25 Rnf8, 38 Rnf168, 39 Rnf4 hypomorphic mice are growth retarded and radiosensitive. In addition, we observed increased cell death and a reduced number of spermatocytes in the seminiferous tubules of these mice.…”

“…In addition, we observed increased cell death and a reduced number of spermatocytes in the seminiferous tubules of these mice. This meiotic defect is also reminiscent of loss of DDR proteins such as H2Ax, 24 Mdc1, 25 Rnf8, 38 and Rnf168, 39 and can be explained by the observation that Spo-11-mediated generation of DSBs and their repair by HR are required for proper meiosis. 37 Together, these data establish an important genetic link between Rnf4 and the DDR in mammalian cells.…”