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. 2012 Apr 18;31(8):1865-78.
doi: 10.1038/emboj.2012.47. Epub 2012 Feb 28.

RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites

Frédérick A Mallette  1 , Francesca Mattiroli, Gaofeng Cui, Leah C Young, Michael J Hendzel, Georges Mer, Titia K Sixma, Stéphane Richard
Affiliations

RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites

Frédérick A Mallette et al. EMBO J. .

Abstract

In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8-dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8- and RNF168-deficient cells to form 53BP1 foci. We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The tandem tudor domains of JMJD2A and JMJD2B bind H4K20(me2) with high affinity. (A) Domain structure of the different members of the JMJD2 family of lysine demethylases and 53BP1. (B) Sequence alignment of the tandem tudor domains of human JMJD2A, JMJD2B, JMJD2C and 53BP1 using TCoffee. Amino acids highlighted in blue show perfect conservation while red denotes amino acids corresponding to functional conservation. (C) Biotinylated peptides immobilized on streptavidin beads were used to pulldown GST-purified JMJD2A and 53BP1 tandem tudor domains. (D) Titrations of JMJD2A-tudor, JMJD2B-tudor and 53BP1-tudor with H4KC20(me2) (residues 12–25). The integrated heat measurements from raw titration data and curve fitting with a standard one-site binding model are shown for each experiment. KD and stoichiometry (n) values are indicated with the associated standard deviations determined by non-linear least squares fitting. The heat of dilution of the H4KC20(me2) peptide is shown in blue, overlaid to the 53BP1-tudor titration signal. Note that the KD of H4KC20(me2) for 53BP1-tudor is about twice that of H4K20(me2).
Figure 2
Figure 2
DNA damage triggers degradation of the lysine demethylase JMJD2A. (A) Immunoblot analysis of the JMJD2 family members in U2OS left untreated or treated with 1 μM doxorubicin (doxo) for 12 h. (B) Protein stability of JMJD2A is affected by IR. U2OS cells were treated with cycloheximide and chloramphenicol for 1 h then exposed to IR (10 Gy). (C) Immunoblot analysis of JMJD2A in U2OS cells exposed to UV (20 J/cm2). (D) Immunoblot analysis of U2OS cells transfected with the empty vector or pLPC Flag–JMJD2A using FUGENE6 and treated with 1 μM doxorubicin for 12 h. (E) JMJD2A protein half-life measurement by immunoblotting in U2OS cells treated or not with 2 μM doxorubicin (doxo) as described in Materials and methods. (F) The quantification of the results of (E) is shown. (G) Protein stability of chromatin-associated JMJD2A in U2OS cells treated with cycloheximide and chloramphenicol for 1 h, and exposed to 2 μM doxorubicin for 60 min.
Figure 3
Figure 3
RNF8 directly ubiquitinates and regulates the stability of JMJD2A. (A) In vivo ubiquitination of JMJD2A in U2OS cells transfected with HA–ubiquitin and Flag–JMJD2A, and treated or not with 5 μM MG132. (B) Protein stability of JMJD2A in U2OS cells pre-treated with the proteasome inhibitor MG132 (5 μM) for 1 h, then treated with cycloheximide and chloramphenicol for 1 h, and exposed to 2 μM doxorubicin for 90 min. (C) JMJD2A protein levels in U2OS cells transfected with plasmids expressing Flag–JMJD2A and increasing amounts of RNF8. GFP was used to monitor transfection efficiency. (D) JMJD2 family members protein levels in U2OS cells transfected with siGFP or siRNAs against RNF8. (E) Relative mRNA levels using RT–qPCR for RNF8 and JMJD2A in U2OS cells transfected with GFP or RNF8 siRNAs using Lipofectamine RNAiMAX. (F) Protein stability of JMJD2A in U2OS cells transfected with siGFP or siRNF8, pre-treated with cycloheximide and chloramphenicol for 1 h and then treated with 2 μM doxorubicin. (G) The Flag–GST-tagged JMJD2A C- and N-terminal regions were used as substrates for ubiquitination by purified RNF8 wild-type or RING domain mutant RNF8I405A in the presence of E1 (Uba1), E2 (UbcH5c) and ubiquitin. Increasing concentrations (0.1, 0.3 or 0.9 μM) of RNF8 or RNF8I405A were used. The ubiquitination reactions were analysed by anti-flag immunoblotting.
Figure 4
Figure 4
RNF8 stimulates the formation of K48-linked ubiquitin chains on JMJD2A. Indirect immunofluorescence of K63- (A) and K48-linked ubiquitin (B) in U2OS cells transfected with Flag–RNF8 or Flag–RNF168. (C) In vivo ubiquitination of JMJD2A in U2OS cells transfected with Flag–JMJD2A, myc–RNF8 and HA–UbWT or the mutant ubiquitin HA-UbK48R, and treated with 5 μM MG132 for 4 h.
Figure 5
Figure 5
RNF168 directly ubiquitinates and regulates JMJD2A protein stability. (A) JMJD2A levels in U2OS cells transfected with plasmids expressing Flag–JMJD2A and RNF8, RNF168 or myc–XIAP. (B) Immunoblots of JMJD2 family members in U2OS cells transfected with siGFP or siRNF168sp. (C) In vitro ubiquitination of JMJD2A by RNF168. The Flag–GST-tagged JMJD2A C- and N-terminal regions were used as substrates for ubiquitination by purified RNF168 wild-type or RING domain mutant RNF168I18A in the presence of E1 (Uba1), E2 (UbcH5c) and ubiquitin. Increasing concentrations (0.1, 0.3 or 0.9 μM) of RNF168 or RNF168I18A were used. The ubiquitination reactions were analysed by anti-flag immunoblotting.
Figure 6
Figure 6
JMJD2A abrogates the recruitment of 53BP1 to DNA damaged sites. (A) Indirect immunofluorescence of 53BP1 and Flag–JMJD2A in U2OS cells transfected with siRNF8 or Flag–JMJD2A and fixed 1 h after irradiation with 3 Gy. Scale bars correspond to 20 μm and arrows indicate transfected cells. (B) Quantification of the γH2AX, MDC1 and 53BP1 foci in (A) and Supplementary Figure S6 is shown. These data represent the average and standard deviation of three independent counts of 100 cells each. (C) Indirect immunofluorescence of RAP80 and Flag–JMJD2A in U2OS cells transfected with siRNF8 or Flag–JMJD2A and fixed 1 h after IR (3 Gy). (D) Indirect immunofluorescence of phosphorylated Chk2 at threonine 68 and flag in U2OS transfected with an empty vector or pLPC Flag–JMJD2A and fixed 1 h after IR (3 Gy). (E) Quantification of the data presented in (F). (F) Indirect immunofluorescence of 53BP1 and flag in U2OS cells transfected with flag-tagged JMJD2A, JMJD2B, JMJD2C or JMJD2D, and fixed 1 h after exposure to IR (3 Gy). Scale bar corresponds to 20 μm and arrows indicate transfected cells.
Figure 7
Figure 7
The integrity of the tandem tudor domain of JMJD2A is required to block 53BP1 foci formation. (A) Indirect immunofluorescence of 53BP1 and flag in U2OS cells transfected with different mutants of Flag–JMJD2A, Flag–JMJD2D or Flag–JARID1C and fixed 1 h after IR (3 Gy). Scale bar corresponds to 20 μm and arrows indicate transfected cells. (B) Quantification of the 53BP1 foci in cells presented in (A). Data represent the average and standard deviation of three independent counts of 100 cells. Asterisk corresponds to a P<0.005 using Student's t-test. (C) Immunoblots for different histone marks and 53BP1 in U2OS cells infected with a retroviral vector encoding Flag–JMJD2A or a control vector.
Figure 8
Figure 8
Combined knockdown of JMJD2A and JMJD2B rescues 53BP1 foci formation in RNF8- and RNF168-depleted cells. (A) Indirect immunofluorescence of 53BP1 in U2OS cells transfected with siRNF8#1 and different combinations of individual siJMJD2A and siJMJD2B, and fixed 1 h after IR (3 Gy). Scale bar corresponds to 20 μm. (B) Quantification of the 53BP1 foci in U2OS cells transfected with siRNF8#1 and different combinations of siJMJD2A and siJMJD2B. These data represent the average and standard deviation of three independent counts of 100 cells. Asterisks correspond to a P<0.05 using Student's t-test. (C) Indirect immunofluorescence of 53BP1 in U2OS cells transfected with a SMARTpool siRNF168sp and different combinations of individual siRNAs targeting JMJD2A and JMJD2B and fixed 1 h after IR (3 Gy). Scale bar corresponds to 20 μm. (D) Quantification of the 53BP1 foci in U2OS cells transfected with siRNF168sp and different combinations of individual siRNAs targeting JMJD2A and JMJD2B. These data represent the average and standard deviation of three independent counts of 100 cells. Asterisks correspond to a P<0.01 using Student's t-test. (E) Colony formation assay of U2OS cells stably infected with a control vector, wild-type or mutated JMJD2A, JMJD2B or JMJD2D and exposed to increasing amounts of etoposide for 3 h. The cells were then incubated for 14 days and stained using crystal violet. Colonies containing >50 cells were counted. The experiment was carried out in triplicate and asterisks represent a P<0.05 using the two-way ANOVA test.
Figure 9
Figure 9
The RNF8- and RNF168-mediated degradation of JMJD2A regulates the formation of 53BP1 foci. Schematic model of the role of RNF8/RNF168 in the degradation of JMJD2A and the formation of 53BP1 and BRCA1/RAP80 foci. In undamaged cells, JMJD2A binds H4K20(me2) via its tandem tudor domain. Following DNA damage, RNF8 initiates the ubiquitination of JMJD2A which is further amplified by RNF168 to ensure efficient and local degradation of JMJD2A leading to the exposure of H4K20(me2). 53BP1, which can bind H4K20(me2) with a lower affinity than JMJD2A/B, can now interact with H4K20(me2) through its tandem tudor to accumulate at sites of DNA damage. RNF8 and RNF168 trigger two types of ubiquitination cascades leading independently to the recruitment of RAP80 and 53BP1.
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References

    1. Acs K, Luijsterburg MS, Ackermann L, Salomons FA, Hoppe T, Dantuma NP (2011) The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks. Nat Struct Mol Biol 18: 1345–1350 - PubMed
    1. Bekker-Jensen S, Danielsen JR, Fugger K, Gromova I, Nerstedt A, Bartek J, Lukas J, Mailand N (2010) HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes. Nat Cell Biol 12: 80–86; sup pp 1–12 - PubMed
    1. Bekker-Jensen S, Lukas C, Melander F, Bartek J, Lukas J (2005) Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1. J Cell Biol 170: 201–211 - PMC - PubMed
    1. Bothmer A, Robbiani DF, Di Virgilio M, Bunting SF, Klein IA, Feldhahn N, Barlow J, Chen HT, Bosque D, Callen E, Nussenzweig A, Nussenzweig MC (2011) Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1. Mol Cell 42: 319–329 - PMC - PubMed
    1. Botuyan MV, Lee J, Ward IM, Kim JE, Thompson JR, Chen J, Mer G (2006) Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127: 1361–1373 - PMC - PubMed

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