Homozygous deficiency of ubiquitin-ligase ring-finger protein RNF168 mimics the radiosensitivity syndrome of ataxia-telangiectasia

Abstract

Maintaining genomic integrity is critical to avoid life-threatening disorders, such as premature aging, neurodegeneration and cancer. A multiprotein cascade operates at sites of DNA double-strand breaks (DSBs) to recognize, signal and repair damage. RNF168 (ring-finger nuclear factor) contributes to this emerging pathway of several E3 ubiquitin ligases that perform sequential ubiquitylations on damaged chromosomes, chromatin modifications essential for aggregation of repair complexes at the DSB sites. Here, we report the clinical and cellular phenotypes associated with a newly identified homozygous nonsense mutation in the RNF168 gene of a patient with a syndrome mimicking ataxia-telangiectasia. The mutation eliminated both of RNF168's ubiquitin-binding motifs, thus blocking progression of the ubiquitylation cascade and retention of repair proteins including tumor suppressors 53BP1 and BRCA1 at DSB sites, consistent with the observed defective DNA damage checkpoints/repair and pronounced radiosensitivity. Rapid screening for RNF168 pathway deficiency was achieved by scoring patients' lymphoblastoid cells for irradiation-induced nuclear foci containing 53BP1, a robust assay we propose for future diagnostic applications. The formation of radiation-induced DSB repair foci was rescued by ectopic expression of wild-type RNF168 in patient's cells, further causally linking the RNF168 mutation with the pathology. Clinically, this novel syndrome featured ataxia, telangiectasia, elevated alphafetoprotein, immunodeficiency, microcephaly and pulmonary failure and has implications for the differential diagnosis of autosomal recessive ataxias.

Figure 1

Analysis of protein levels of selected DDR factors and radiosensitivity of RS66 cells. (a) Cell lysates from RS66 and control (Choc26) cells were analyzed by immunoblotting with the indicated antibodies (see Materials and Methods section). The red box highlights the lack of RNF168 protein in RS66 cells. (b) Colony survival assay demonstrates AT-like radiosensitivity of RS66 cells. Asterisk refers to SF% ranges defined in Sun et al. The color reproduction of this figure is available on the html full text version of the manuscript

Figure 2

Functional analysis of DDR in RS66 cells. (a) RS66 cells exhibit an intra-S phase cell cycle checkpoint defect similar to A-T cells. WT, A-T and RS66 LCLs were labeled with ¹⁴C thymidine for 24 h, irradiated with the doses indicated, and then chased with ³H thymidine (H³T) for 1 h. Cells were fixed and the H³T/¹⁴C ratio, indicative of S-phase inhibition, was calculated by using a scintillation counter. Error bars represent the mean±S.D. N=3. (b) RS66 cells maintain increased levels of phosphorylated SMC1s966 24 h post-IR. RS66 LCLs were mock-irradiated or exposed to 10 Gy of IR and fixed at the times indicated after IR (bottom). WT (top) and A-T (middle) LCLs controls were included in each experiment. Nuclear extracts were prepared and subjected to SDS-PAGE/western blot analysis using the antibodies indicated

Figure 3

Immunocytochemical analysis of 53BP1 and BRCA1 foci formation in response to ionizing radiation. (a) 53BP1 IRIF formation. RS66 and control (Choc26) cells were exposed to 3 Gy of IR and 1 h later immunostained with the antibodies to 53BP1 and gamma-H2AX. The red-framed field shows cells with impaired focal accumulation of 53BP1. (b) BRCA1 IRIF formation. RS66 and control (Choc26) cells were treated as in (a) and immunostained with antibodies to BRCA1 and MDC1. The green-framed field shows cells with impaired focal accumulation of BRCA1

Figure 4

Identification of the RNF168 gene mutation in RS66, and phenotypic rescue of DNA damage signaling by wild-type RNF168 in the RS66 cells. (a) Sequence of genomic DNA from RS66 cells, showing a homozygous nonsense mutation at c.391C>T (R131X) (shaded column). (b) 53BP1 and BRCA1 foci formation in RS66 cells reconstituted by ectopic wild-type RNF168. RS66 cells were transfected with the control plasmid coding for free nuclear GFP (nucGFP, upper row) or the expression plasmid for wild-type GFP-RNF168 (bottom row). The productively transfected cells were identified according to the nuclear GFP signal (green channel) and scored for 53BP1 (left panel) or BRCA1 (right panel) focus formation. Red arrows (upper row) point to control cells lacking nuclear foci for either 53BP1 or BRCA1; green arrows (lower row) mark RNF168-complemented cells that regained the ability to accumulate 53BP1 and BRCA1 in nuclear foci. The merged images contain cells with yellow foci (red on a green background)