Alternative titles; symbols
HGNC Approved Gene Symbol: RNF8
Cytogenetic location: 6p21.2 Genomic coordinates (GRCh38) : 6:37,353,983-37,394,734 (from NCBI)
RNF8 is an E3 ubiquitin ligase that interacts with the E2 ubiquitin-conjugating enzyme UBC13 (UBE2N; 603679) and catalyzes formation of lys63-linked ubiquitin chains on histone H2A (see 613499) flanking sites of DNA damage. This ubiquitination is required to recruit downstream repair and signaling factors to damaged DNA (summary by Bekker-Jensen et al. (2010)).
By sequencing clones from a size-fractionated brain cDNA library, Ishikawa et al. (1998) cloned RNF8, which they designated KIAA0646. The cDNA contains repetitive elements in its 3-prime end. The deduced 485-amino acid protein contains a C3HC4-type zinc finger signature, suggesting that it is involved in nucleic acid management. RT-PCR analysis detected moderate RNF8 expression in brain, testis, and ovary, with little to no expression in all other tissues examined. In vitro translation resulted in a protein with an apparent molecular mass of 62 kD by SDS-PAGE.
Using oligo capping, Seki et al. (1998) cloned RNF8 from a neuroblastoma cDNA library. The deduced 485-amino acid protein has a calculated molecular mass of 56 kD. RT-PCR analysis detected variable expression in all human tissues examined except spleen.
Using yeast 2-hybrid analysis and protein pull-down assays, Takano et al. (2004) showed that RNF8 and retinoid X receptor-alpha (RXRA; 180245) interacted via their N-terminal regions. Overexpression of RNF8 in COS-7 cells resulted in interaction and colocalization of RNF8 and RXRA in the nucleus. A point mutation in RNF8 that disrupted the RING finger or deletion of the N-terminal region of RNF8 prevented localization of RNF8 to the nucleus without affecting nuclear localization of RXRA. Transient overexpression of RNF8 enhanced RXRA-mediated transactivation of an RXR-responsive element (RXRE) in a dose-dependent and retinoic acid-independent manner and upregulated expression of genes downstream of RXRE. Enhancement of transactivation was not seen with RNF8 carrying the RING finger-disrupting mutation or the N-terminal deletion. Takano et al. (2004) concluded that RNF8 is a regulator of RXRA-mediated transcriptional activity.
Kolas et al. (2007) determined that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 (605230) and BRCA1 (113705) focal accumulation at sites of DNA lesions. Moreover, Kolas et al. (2007) established that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA damage-activated protein kinase ataxia telangiectasia mutated (ATM; 607585). They showed that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. RNF8 was also shown to promote the G(2)/M DNA damage checkpoint and resistance to ionizing radiation. Kolas et al. (2007) concluded that their results demonstrated how the DNA damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.
Bekker-Jensen et al. (2010) found that the putative E3 ubiquitin ligase HERC2 (605837) was essential to recruit UBC13 to RNF8 and initiate histone H2A polyubiquitination at sites of DNA damage. Association of HERC2 with RNF8 increased following exposure of HEK293T cells to ionizing radiation. HERC2 also interacted in a ternary complex with MDC1 and RNF8, and all 3 proteins accumulated at sites of DNA double-strand breaks (DSBs). Depletion of HERC2 in HEK293T cells did not impair accumulation of RNF8 and MDC1 at sites of DNA damage, but it caused failure to recruit UBC13 to RNF8, leading to failure of histone H2A polyubiquitination and assembly of downstream DNA damage repair and signaling factors. Bekker-Jensen et al. (2010) hypothesized that HERC2 may stabilize the interaction of RNF8 with UBC13 or inhibit the interaction of RNF8 with other E2 enzymes with which it interacts.
Yan et al. (2012) noted that RNF8 works with UBC13 to catalyze lys63-linked polyubiquitination of H2A (see 613499)-type histones at sites of DNA damage. They found that depletion of RNF8 in HeLa cells abrogated the recruitment of FAAP20 (C1OR86; 615183) to sites of DNA damage. Knockdown of either RNF8 or FAAP20 abrogated the recruitment of FANCA (607139) and FANCD2 (613984) to sites of DNA damage.
Orthwein et al. (2014) unraveled how mitosis blocks DNA DSB repair and determined the consequences of repair reactivation. Mitotic kinases phosphorylate the E3 ubiquitin ligase RNF8 and the nonhomologous end-joining factor 53BP1 to inhibit their recruitment to DSB-flanking chromatin. Restoration of RNF8 and 53BP1 accumulation at mitotic DSB sites activates DNA repair but is, paradoxically, deleterious. Aberrantly controlled mitotic DSB repair leads to Aurora kinase B (AURKB; 604970)-dependent sister telomere fusions that produce dicentric chromosomes and aneuploidy, especially in the presence of exogenous genotoxic stress. Orthwein et al. (2014) concluded that the capacity of mitotic DSB repair to destabilize the genome explains the necessity for its suppression during mitosis, principally due to the fusogenic potential of mitotic telomeres.
Thorslund et al. (2015) elucidated how RNF8 and UBC13 (603679) promote recruitment of RNF168 (612688) and downstream factors to DSB sites in human cells. Thorslund et al. (2015) established that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identified histone H1 as a key target of RNF8-UBC13 in DSB signaling and expanded the concept of the histone code by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance.
Using radiation hybrid analysis, Ishikawa et al. (1998) mapped the RNF8 gene to chromosome 6. Seki et al. (1998) mapped the RNF8 gene to chromosome 6p21.3 by somatic cell hybrid and radiation hybrid analyses.
Santos et al. (2010) reported that Rnf8 -/- mice were defective in class switch recombination (CSR) and accumulated immunoglobulin heavy chain (IgH; see 147100)-associated double-strand breaks (DSB). The CSR DSB repair defect was milder than that in 53bp1 -/- mice, but similar to that in H2ax (601772) -/- mice. Like H2ax -/- mice, Rnf8 -/- males were sterile, and this was associated with defective XY chromatin ubiquitylation. Mice lacking both H2ax and Rnf8 did not have further impairment of CSR. Although 53bp1 foci formation is Rnf8 dependent, 53bp1 could bind to chromatin in the absence of Rnf8. Santos et al. (2010) proposed that there is a 2-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage-inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.
Li et al. (2010) studied Rnf8 -/- mice and observed growth retardation, reduced hematopoietic populations, impaired spermatogenesis, impaired CSR, and increased sensitivity to ionizing radiation in vitro and in vivo. Activated Rnf8 -/- B cells were able to recruit reduced amounts of 53bp1 via Rnf8-independent mechanisms. Rnf8 -/- mice also had a somewhat increased cancer predisposition. Li et al. (2010) concluded that RNF8 is a tumor suppressor whose loss also results in increased non-cancer-related mortality, likely due to immunodeficiency.
Bekker-Jensen, S., Rendtlew Danielsen, J., Fugger, K., Gromova, I., Nerstedt, A., Lukas, C., Bartek, J., Lukas, J., Mailand, N. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes. Nature Cell Biol. 12: 80-86, 2010. Note: Erratum Nature Cell Biol. 12: 412 only, 2010. [PubMed: 20023648] [Full Text: https://doi.org/10.1038/ncb2008]
Ishikawa, K., Nagase, T., Suyama, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., Ohara, O. Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res. 5: 169-176, 1998. [PubMed: 9734811] [Full Text: https://doi.org/10.1093/dnares/5.3.169]
Kolas, N. K., Chapman, J. R., Nakada, S., Ylanko, J., Chahwan, R., Sweeney, F. D., Panier, S., Mendez, M., Wildenhain, J., Thomson, T. M., Pelletier, L., Jackson, S. P., Durocher, D. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318: 1637-1640, 2007. [PubMed: 18006705] [Full Text: https://doi.org/10.1126/science.1150034]
Li, L., Halaby, M.-J., Hakem, A., Cardoso, R., El Ghamrasni, S., Harding S., Chan, N., Bristow, R., Sanchez, O., Durocher, D., Hakem, R. Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer. J. Exp. Med. 207: 983-997, 2010. [PubMed: 20385750] [Full Text: https://doi.org/10.1084/jem.20092437]
Orthwein, A., Fradet-Turcotte, A., Noordermeer, S. M., Canny, M. D., Brun, C. M., Strecker, J., Escribano-Diaz, C., Durocher, D. Mitosis inhibits DNA double-strand break repair to guard against telomere fusions. Science 344: 189-193, 2014. [PubMed: 24652939] [Full Text: https://doi.org/10.1126/science.1248024]
Santos, M. A., Huen, M. S. Y., Jankovic, M., Chen, H.-T., Lopez-Contreras, A. J., Klein, I. A., Wong, N., Barbancho, J. L. R., Fernandez-Capetillo, O., Nussenzweig, M. C., Chen, J., Nussenzweig, A. Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8. J. Exp. Med. 207: 973-981, 2010. [PubMed: 20385748] [Full Text: https://doi.org/10.1084/jem.20092308]
Seki, N., Hattori, A., Sugano, S., Suzuki, Y., Nakagawara, A., Ohhira, M., Muramatsu, M., Hori, T., Saito, T. Isolation, tissue expression, and chromosomal assignment of a novel human gene which encodes a protein with RING finger motif. J. Hum. Genet. 43: 272-274, 1998. [PubMed: 9852682] [Full Text: https://doi.org/10.1007/s100380050088]
Takano, Y., Adachi, S., Okuno, M., Muto, Y., Yoshioka, T., Matsushima-Nishiwaki, R., Tsurumi, H., Ito, K., Friedman, S. L., Moriwaki, H., Kojima, S., Okano, Y. The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity. J. Biol. Chem. 279: 18926-18934, 2004. [PubMed: 14981089] [Full Text: https://doi.org/10.1074/jbc.M309148200]
Thorslund, T., Ripplinger, A., Hoffmann, S., Wild, T., Uckelmann, M., Villumsen, B., Narita, T., Sixma, T. K., Choudhary, C., Bekker-Jensen, S., Mailand, N. Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage. Nature 527: 389-393, 2015. [PubMed: 26503038] [Full Text: https://doi.org/10.1038/nature15401]
Yan, Z., Guo, R., Paramasivam, M., Shen, W., Ling, C., Fox, D., III, Wang, Y., Oostra, A. B., Kuehl, J., Lee, D.-Y., Takata, M, Hoatlin, M. E., Schindler, D., Joenje, H., de Winter, J. P., Li, L., Seidman, M. M., Wang, W. A ubiquitin-binding protein, FAAP20, links RNF8-mediated ubiquitination to the Fanconi anemia DNA repair network. Molec. Cell 47: 61-75, 2012. [PubMed: 22705371] [Full Text: https://doi.org/10.1016/j.molcel.2012.05.026]