Cells respond to cytotoxic DNA double-strand breaks (DSBs) by recruiting DNA repair proteins to the damaged site. This recruitment is dependent on ubiquitylation of adjacent chromatin areas by E3 ubiquitin ligases such as RNF8 and RNF168, which are recruited sequentially to the DSBs. However, it is unclear what dictates the sequential order and recruits RNF168 to the DNA lesion. Here, we reveal that L3MBTL2 (lethal(3)malignant brain tumour-like protein 2) is the missing link between RNF8 and RNF168. We found that L3MBTL2 is recruited by MDC1 and subsequently ubiquitylated by RNF8. Ubiquitylated L3MBTL2, in turn, facilitates recruitment of RNF168 to the DNA lesion and promotes DNA DSB repair. These results identify L3MBTL2 as a key target of RNF8 following DNA damage and demonstrates how the DNA damage response pathway is orchestrated by ubiquitin signalling.
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We thank Z. Zhang, M. Jasin, X. Yu, D. Reinberg, N. Dantuma and N. Mailand for providing reagents. Thank you to the members of the Lou lab for helpful discussions and B. Davies for technical assistance. This work was supported, in part, by NIH grants CA130996, CA108961 and CA148940 (to Z.L.) and CA160333 (to Y.D.). S.N. was supported by the Laura J. Siegel Breast Cancer Fellowship Award from the Foundation for Women’s Wellness. K.A. and S.N. thank the Mayo Clinic MSTP for fostering an outstanding environment for physician-scientist training. We thank the Mayo Clinic Cancer Center for the use of the Cytogenetics Core, which provided PNA FISH services. The Mayo Clinic Cancer Center is supported in part by an NCI Cancer Center Support Grant 5P30 CA15083-36.
The authors declare no competing interests.
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Integrated supplementary information
. (a-b) The specificity of the L3MBTL2 antibody was validated by (a) western blot and (b) immunofluorescence. Wild-type or L3MBTL2 knockout U2OS cells were exposed to 2GY irradiation, fixed after an hour, and stained for L3MBTL2 (red). Nucleus was stained with DAPI (blue). Shown are representative images from 3 independent experiments in b. Scale bars, 10 μm. Representative western blots are provided from 3 biologically independent experiments in a. Unprocessed western blots are provided in Supplementary Figure 5
. (a-c) G9A knockdown does not affect RNF168 focus formation in U2OS cells. (d-f) E2F6 knockdown does not affect RNF168 focus formation in U2OS cells. (g-i) PCGF6 knockdown does not affect RNF168 focus formation in U2OS cells. (j-l) L3MBTL1 knockdown does not affect RNF168 focus formation in U2OS cells. (m) MDC1, RNF8 and RNF168 interact with endogenous L3MBTL2 following DNA damage. (n) Knockdown of RNF168 using shRNA does not affect the interaction between RNF8 and L3MBTL2. (o) Knockdown of RNF8 affects the interaction between RNF168 and L3MBTL2. (p) The FHA mutant of MDC1 (R58A) is unable to interact with L3MBTL2 upon DNA damage. The indicated constructs were transfected into MDC1 knockout cells. Cells were exposed to the indicated doses of irradiation. Lysates were collected after an hour and the interaction between RNF168 and L3MBTL2 was assessed. (q) The phosphorylation mutant of L3MBTL2 does not interact with RNF168 upon DNA damage. The indicated constructs were transfected into L3MBTL2 knockout cells. Cells were exposed to the indicated doses of irradiation. Lysates were collected after an hour and the interaction between RNF168 and L3MBTL2 was assessed. (r-t) RNF8 is dispensable for radiation-induced L3MBTL2 foci formation. RNF8 was knocked out using shRNA (GFP-tagged). Cells were exposed to 2GY irradiation, fixed after an hour, and L3MBTL2 foci (red) formation was assessed. Scale bars, 10 μm. Data are represented as the mean ± SEM of n = 3 independent experiments in b, e, h, k and s. Circles depict individual data points. Statistical significance was calculated using 2-way ANOVA. **p = 0.0001 and p=0.001 for control shRNA vs RNF8 shRNA1 and shRNA2 with IR, respectively. Representative western blots are provided from 3 biologically independent experiments in c, f, i, l-q and t. The experiments in a, d, g, j, and r were repeated 3 independent times with similar results. Source data and unprocessed western blots are provided in Supplementary Table 1 and Supplementary Figure 5, respectively
Supplementary Figure 3 RNF8 mediated K63 linked ubiquitylation of L3MBTL2 following DNA damage is critical for the interaction with UDM1 of RNF168 and subsequent histone ubiquitylation.
. (a) Endogenous L3MBTL2 is ubiquitylated by RNF8. Knockdown of RNF8 with shRNA abrogates this DNA damage induced ubiquitylation of L3MBTL2. (b) MDC1 facilitates DNA damage induced L3MBTL2 ubiquitylation. (c) The phosphorylation mutant of L3MBTL2 (S335A) cannot be efficiently ubiquitylated following DNA damage. (d-f) Knockdown of Histone H1 does not affect RNF168 foci formation following DNA damage. (g-i) Comparison of the effect of histone H1 and L3MBTL2 knockdown on various DNA repair proteins. (j-l) HMGB1 does not affect RNF168 foci formation. (m) Histone H1 is mono ubiquitylated following DNA damage. Cells were transfected with the indicated plasmids and exposed to radiation. Lysates were collected after an hour and ubiquitylation of histone H1 was assessed. USP2 was added as indicated and deubiquitylation of histone H1 was assessed to validate that the band observed is indeed histone H1. (n) Histone H1 does not interact with UDM1 following DNA damage. Cell lysates from irradiated cells were incubated with GST-tagged UDM1. Proteins were eluted and blots were probed with the indicated antibodies. (o) H1.x variant of histone H1 does not recognize K63 UIM following DNA damage in MDA-MB-231 cells. (p) Cells were irradiated to induce DNA damage, lysed after 1 hour, and reciprocal endogenous IP was performed with L3MBTL2 and Histone H1 antibodies. No interaction between L3MBTL2 and Histone H1 was observed. Scale bars, 10 μm. Experiments in e and j were repeated 3 independent times with similar results. Data are represented as the mean ± SEM of n = 3 independent experiments in d, i and k. Circles depict individual data points. Statistical significance was calculated using 1-way ANOVA in d and k and 2-way ANOVA in panel i. **p = 0.001 for control vs Histone H1 siRNA with IR in d. **p = 0.000000001 (BRCA1) and p = 0.000000000000007 (53BP1) for control siRNA vs L3MBTL2 siRNA with IR in i. *p = 0.0117 (53BP1) for control siRNA vs Histone H1 siRNA with IR in i. Representative western blots are provided from 3 biologically independent experiments in a-c, f-h, and l-p. Source data and unprocessed western blots are provided in Supplementary Table 1 and Supplementary Figure 5, respectively
Supplementary Figure 4 DNA damage induced RNF8 mediated ubiquitylation of L3MBTL2 is critical for DNA DSB repair.
. (a) Representative images showing RNF168 foci formation in U2OS cells expressing the indicated plasmids (related to Fig. 5e). (b-c) L3MBTL2 knockout or ubiquitylation mutant does not affect protein levels. (d) Representative blot showing the knockdown and knockout efficiency of RNF168 and L3MBTL2 in MDA-MB-231 cells (related to Fig. 5g). (e-f) Loss of L3MBTL2 sensitizes cells to irradiation. (e) Representative blot showing expression levels of exogenous wild-type (WT) or ubiquitylation mutant (K659R) L3MBTL2 in MDA-MB-231 cells. (f) Survival plot of L3MBTL2 knockout MDA-MB-231 cells expressing the indicated plasmids at endogenous levels when exposed to the indicated doses of radiation. (g-h) Loss of L3MBTL2 sensitizes cells to irradiation. (g) Representative blot showing the knockdown and knockout efficiency of RNF168 and L3MBTL2 in U2OS cells. (h) Survival assays of L3MBTL2 knockout U2OS cells treated as indicated and exposed to the indicated doses of irradiation. (i-j) Loss of L3MBTL2 reduces radiation-induced FK2 foci formation. (i) Representative blot showing the knockout efficiency of L3MBTL2. (j) Representative images showing FK2 foci formation in L3MBTL2 control and knockout U2OS cells. (k-l) The MBT domains of L3MBTL2 are important for its role in DNA damage response. (k) Deletion of any of the MBT domains disrupts RNF168 foci formation. (l) Expression level of the various L3MBTL2 constructs utilized in (k). Scale bars, 10 μm. Experiments in a and j were repeated 3 independent times with similar results. Data in f, h, and k are represented as the mean ± SEM of n = 3 independent experiments. Circles depict individual data points. Statistical significance was calculated using 2-way ANOVA in f and h and 1-way ANOVA in panel k. **p = 0.0001 for vector vs all other groups except L3MBTL2 sgRNA + Flag L3MBTL2 WT at each time point in f and h. **p = 0.0000000000001 for WT vs all other MBT deletion groups and p = 0.0004 for WT vs ∆ZN in k. Representative western blots are provided from 3 biologically independent experiments in b-e, g, i and l. Source data and unprocessed western blots are provided in Supplementary Table 1 and Supplementary Figure 5, respectively
. Unprocessed images for all blots in this manuscript. Unprocessed images for all blots in this manuscript
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Nowsheen, S., Aziz, K., Aziz, A. et al. L3MBTL2 orchestrates ubiquitin signalling by dictating the sequential recruitment of RNF8 and RNF168 after DNA damage. Nat Cell Biol 20, 455–464 (2018). https://doi.org/10.1038/s41556-018-0071-x
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