In this study, we demonstrated that Jab1 is essential for the early development of mouse embryos. Loss of Jab1 activity through targeted disruption of the gene in the germ line led to the arrest of embryonic development soon after implantation. Our data is supported by the work published by Tomoda et al. (2004)
which showed that loss of Jab1 resulted in early embryonic lethality in mice, and is also supported by recent CSN knockout and mutational studies in a variety of organisms (Doronkin et al., 2002
; Suh et al., 2002
; Lykke-Andersen et al., 2003
; Tomoda et al., 2004
; Harari-Steinberg et al., 2007
). In Arabidopsis thaliana
, for example, mutations in the various subunits of the CSN resulted in severely retarded development of seedlings and lethality after the seedling stage (Kwok et al., 1998
); in Drosophila melanogaster
, loss of CSN caused a complex phenotype that included defects in hematopoiesis, axonal guidance, and steroid hormone signaling (Oron et al., 2007
); and in fission yeast, the CSN complex was shown to be important for cell cycle control (Mundt et al., 1999
). Also, embryonic lethality was observed when CSN5 occurred at the same developmental stage in CSN2 and CSN3 knockout mice (Lykke-Andersen et al., 2003
; Yan et al., 2003
). Together, these studies showed embryonic lethality occurring at the developmental stage in mice, as has been the case with other organisms. However, the cause of embryonic death is not fully understood. Our data presented here revealed that defects in HR of DSB repair by reduction of DNA repair protein Rad51 may be one of major mechanisms for embryonic death in Jab1-deficient mice.
The structural integrity of DNA is continually being challenged by the presence of chemical and physical factors in the environment. In addition to lesions caused by exogenous agents, DNA undergoes spontaneous decay, replication errors, and other types of damage resulting from normal metabolic processes (Lindahl, 1993
). Repair of damaged DNA is therefore crucial for maintenance of genomic integrity and cell survival (Jackson and Bartek, 2009
). In our studies, in the absence of stimulation of exogenous DNA damage, Jab1-deficient embryos and osteosarcoma cells showed increased incidence of a spontaneous genome instability phenotype, including a large number of TUNEL foci in Jab1−/−
embryos and blastocysts and an increased number of γ-H2AX foci with a decreased percentage of intact DNA in Jab1-deficient MEFs and human U2OS cells. These findings suggested that Jab1-deficient cells promote spontaneous DNA breaks; therefore, loss of Jab1 may affect efficient DNA repair.
DSBs are the most lethal form of DNA damage (Jackson and Bartek, 2009
). DSB can either be properly repaired, restoring genomic integrity, or misrepaired, resulting in drastic consequences, such as cell death, genomic instability, and cancer (Jackson and Bartek, 2009
). A number of methods for repairing DSBs have evolved in mammalian cells, and one of the most important is HR, which exists predominantly in the S-phase during DNA replication. Because it is error-free, HR plays a critical role in genome maintenance and cell survival (Khanna and Jackson, 2001
; van Gent et al., 2001
). In eukaryotes, the DNA-repairing protein Rad51 has a central function in HR of DSB repair by forming nucleoprotein filaments and mediating strand exchange between DNA duplexes (Shinohara et al., 1992
; Haaf et al., 1995
). Rad51 is essential for embryonic survival in response to exogenous DNA-damaging agents (Tsuzuki et al., 1996
) and for the repair of spontaneously occurring chromosome breaks in proliferating cells (Sonoda et al., 1998
). Our results that increased number of spontaneous DNA breaks were correlated with reduced expression of Rad51 indicated that DNA repair defect was the cause of increased spontaneous DNA breaks in Jab1-deficient cells. This could explain the embryonic lethality in Jab1 knockout mice and the impaired growth capacity and increase cell death in Jab1+/−
MEFs. In Jab1 knockdown cells, the reduced Rad51 levels and the uninhibited expression of Ku70, a key protein in the NHEJ DNA-repair pathway, led us to believe that Jab1-affected DNA repair may occur through HR and may not affect the NHEJ DNA-repair pathway.
How Jab1 regulates Rad51 protein and mRNA expression is not completely understood, but it is clear that Jab1 affects the major regulator of Rad51, p53, which was detected in high levels in Jab1−/−
embryos, Jab1-deficient MEFs, and Jab1 knockdown U2OS cells in our study; furthermore, these results have been supported by several other studies that found that JAB1 contributes to the stability of Mdm2 and accelerates p53 degradation (Bech-Otschir et al., 2001
; Oh et al., 2006
) and that p53 negatively regulates Rad51 at the transcriptional level through a p53-response element (Arias-Lopez et al., 2006
; Hannay et al., 2007
). Our data provided new evidence that the inhibition of Jab1 directly affects p53-binding activity in vivo
and in vitro
, resulting in deregulation of the Rad51 level and defects in HR DNA repair.
Human recombinase Rad51 is a key protein for the maintenance of genome integrity and for cancer development (Shinohara et al., 1992
; Gupta et al., 1997
). As with Jab1, increased expression of Rad51 has been reported in various types of malignant tumors, including breast, lung, and pancreatic adenocarcinoma (Maacke et al., 2000
). In addition, aberrant amounts of Rad51 have been observed in a number of transformed cell types that may induce malignant transformation (Xia et al., 1997
; Raderschall et al., 2002
). Furthermore, overexpression of Rad51 can enhance spontaneous recombination frequency and increase resistance to DSB-inducing cancer therapies (Vispe et al., 1998
; Hannay et al., 2007
Many chemotherapeutic drugs have been used to kill proliferating cells, causing extensive DNA damage that ultimately leads to cell cycle arrest and cell death. However, the efficacy of these therapeutic agents can be significantly reduced by the ability of cells to repair DNA. The inhibition of Rad51, therefore, has been explored as a way to sensitize cancer cells to chemotherapy and radiotherapy (Collis et al., 2001
; Russell et al., 2003
). Our finding that reduced Rad51 levels by Jab1 deficiency suggests the possibility that inhibiting Jab1 in cancer cells may enhance sensitivity of these cells to DNA-damaging chemotherapeutic agents and/or IR. Inhibition of Jab1 not only blocks cancer cell proliferation, but also reduces the DNA HR repair function after cancer therapy. The association between Jab1 and Rad51 inhibition and drug and IR therapeutic resistance should be further studied.
In conclusion, we demonstrated that defects in DSB repair in Jab1 gene–targeted embryos contribute to premature cell death in homozygous Jab1 knockout mice. We believe that accumulated p53 strongly inhibits Rad51 promoter activity and acts as a major mechanism for HR repair defects in Jab1-deficient cells and embryos. These findings will contribute to the understanding of the overall mechanisms of Jab1 regulation in cell proliferation, DNA repair, and cell survival. Jab1-deficient cells and animal models may clarify the role of Jab1 in the regulation of target molecules in cancer, such as p53 and Rad51, and the abnormalities that accompany their dysregulation, establishing Jab1 as a potential target for cancer therapy in humans.