Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation

Abstract

Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of γH2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity.

Keywords: DNA damage; DNA damage response; histone modification; signal transduction; ubiquitylation (ubiquitination).

Figure 1.

Identification of a negative regulator of 53BP1 foci formation. A, schematic of the screen used in this study. B, the indicated RNF protein expression vectors were transfected into 293T cells. After 48 h, the transfected 293T cells were exposed to 10 Gy of ionizing radiation. Four hours after the irradiation, the cells were fixed and stained with anti-53BP1 antibody. DAPI was used as a nuclear indicator. The results represent the average of two independent experiments. Error bars indicate the standard deviation. C, Knockdown of RNF126 by transfection with siRNA against RNF126. Control or RNF126 siRNA #1-, #2-, or #3-transfected cell lysates were analyzed by SDS-PAGE and immunoblotting with the specified antibodies. D, 293T cells were transfected with control or RNF126 siRNA #1. After 48 h, transfected 293T cells were exposed to 2 Gy of ionizing radiation. After 0, 1, 6, or 24 h, cells were fixed and stained with anti-53BP1 antibody. E, U2OS cells were transfected with control or siRNF and then treated with the ATM inhibitor KU55933 for 1 h at 5 μm. Cells were then irradiated at 1 Gy. Immunofluorescence detection of 53BP1 foci was observed 8 h after irradiation, and statistics was analyzed.

Figure 2.

Identification of RNF126 regions to inhibit 53BP1 focus formation. A, C, and E, diagrams of WT RNF126, serial deletion mutants, and point mutation constructs. B, D, and F, RNF126 inhibits 53BP1 focus formation through its ZF, central region, and RING domains. 293T cells were transfected with plasmids encoding WT RNF126 and serial deletion mutants. After 48 h, the transfected 293T cells were exposed to 10 Gy of ionizing radiation. Four hours after irradiation, the cells were fixed and stained with anti-53BP1 antibody. The results represent the average of two independent experiments. Error bars indicate the standard deviation for each expression plasmid–transfected cell.

Figure 3.

RNF126 regulates recruitment of repair proteins to DSBs. A, schematic of the IR-induced DNA damage signaling cascade. B, 293T cells were transfected with the GFP-RNF126 expression plasmid. After 48 h, the transfected 293T cells were exposed to 10 Gy of ionizing radiation. Four hours after irradiation, the cells were fixed and stained with an anti-γH2AX, -MDC1, anti-FK2, anti-RAP80, anti-BRCA1, or anti-53BP1 antibody. DAPI was used as a nuclear indicator. The results represent the average of two independent experiments. Error bars indicate the standard deviation for each expression plasmid–transfected cell. C, Myc-RNF8 or Myc-RNF168 with/without the GFP-RNF126 plasmid was transfected into 293T cells. After 48 h, transfected 293T cells were exposed to 10 Gy of ionizing radiation. Four hours after irradiation, the cells were fixed and stained with an anti-Myc antibody. DAPI was used to indicate the nuclei. The results represent the average of two independent experiments. Error bars indicate the standard deviation for each expression plasmid–transfected cell.

Figure 4.

RNF126 regulates H2A monoubiquitination. A, C, and E, 293T cells were transfected with the indicated expression vectors. After 48 h, the transfected 293T cells were fractionated into soluble and chromatin fractions and then subjected to immunoblot analysis with the indicated antibodies. WB, Western blot. B, diagram of SFB-H2A point mutants. D, 293T cells were transfected with a combination of siRNA and expression vectors. After 48 h, the transfected 293T cells were fractionated into soluble and chromatin fractions and then subjected to immunoblot analysis with the indicated antibodies. ctrl, control. F, 293T cells were transfected with the indicated expression vectors. After 48 h, transfected 293T cells were exposed to 10 Gy of ionizing radiation. One hour after irradiation, the cell lysates were pulled down with streptavidin beads and then subjected to the indicated antibodies.

Figure 5.

Subcellular localization of RNF126 in response to DNA damage. A, U2OS cells were transfected with GFP-RNF126 fusion protein expression vectors, and after 48 h, cells were treated with laser microirradiation. After 10 min, the cells were fixed and stained with anti-γH2AX. DAPI was used to indicate the nuclei. B, U2OS cells were treated with laser microirradiation. The cells were fixed and stained with anti-RNF126 and -γH2AX. The results represent the average of three independent experiments. Error bars indicate the standard deviation. C, mCherry-LacI-FokI was transfected with the indicated expression vectors into U2OS-DSB reporter cells. After 48 h, live-cell imaging was performed by confocal microscopy. D, the kinetics of GFP-RNF126 translocation to sites of DNA damage. E and F, GFP-RNF126 translocation to DNA damage sites is dependent on its ZF domain. The results represent the average of three independent experiments. Error bars indicate the standard deviation. G, GFP-RNF126 NLS-ZF translocation to sites of DNA damage.

Figure 6.

RNF126 is required for DNA damage repair. A and B, U2OS cells harboring the NHEJ reporter system were transfected with the indicated expression vectors. After 24 h, the cells were transfected again together with the I-SceI expression vector. After 72 h of incubation, the GFP expression level was analyzed by flow cytometry.