RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response

Abstract

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

Figure 1. DNA binding motif within hRNF4 RING domain

A Sequence alignment of human RNF4 RING domain with the homologous regions of RNF168 and RING1b. B Structural superimposition of the basic RKK cluster of hRNF4 RING domain (cyan) with the homologous RRR cluster in RNF168 (green) and RING1b (salmon); zinc atoms are shown as gold spheres. C DNA docking (DOT)-based molecular docking studies on the RNF4:UbcH5a:ubiquitin trimeric complex crystal structure (4AP4.pdb) with 12-bp DNA, RNF4 RING domains in cyan, ubiquitin in blue and UbcH5a E3 in green. The 2,000 most favorable interactions denoted by red spheres, with the R₁₇₇-K_178-K₁₇₉ basic cluster in the C-terminus of the RNF4 RING domain highlighted in yellow, and top scoring dsDNA predictions depicted as cartoon images in orange. D Left panel: GASBOR space fill model of the in-solution small angle X-ray scattering (SAXS) structure of apo-hRNF4, into which the hRNF4 RING crystal structure (2XEU.pdb) is fitted and illustrated as a cartoon diagram. Right panel: MONSA-based calculation of the in-solution SAXS shape of hRNF4 (cyan) in complex with a 12-bp dsDNA oligonucleotide (orange), depicted as dummy beads.

Figure 2. DNA binding and E3 Ligase activity of RNF4-K179D

A GST-hRNF4 RING_120–190aa domain purified using biotin-labeled duplex DNA, detected with an anti-GST antibody. Wild-type (WT) RNF4 RING domain, mutant K179D (KD) and free GST are shown. B Kinetics of di-ubiquitin species formation by GST-RNF4. Mean and s.d. scored from triplicate experiments. C Anti-FLAG western of in vitro ubiquitination assay in the presence of 12-bp duplex DNA titrated up to 150-fold molar excess relative to GST-RNF4 (0.2 μM). D Anti-FLAG and anti-H3 western blot of in vitro ubiquitination of recombinant (H3) versus nucleosomal (N) histone H3 by wild-type (WT) and K179D mutant (KD) GST-RNF4 RING domain. E Quantitation of unmodified H3 relative to free H3 and nucleosomal H3 input [lanes 1 and 2, respectively, in anti-H3 blot of (D)]. Mean and s.d. scored from triplicate experiments.

Figure 3. RNF4 is required for DNA damage response at telomeres

A Metaphase spreads derived from mouse embryonic fibroblasts infected with the indicated constructs and either depleted for TRF2 (+OHT) or untreated (-OHT) were stained for telomeric DNA (green) and with DAPI (red). Percentages of fused chromosome ends are indicated. B Telomeric restriction analysis performed on genomic DNA derived from cells treated as in (A). Hybridization in native conditions shows single-stranded telomeric DNA (Native). Hybridization following denaturation shows total telomeric DNA (Total). C Quantitation of signal in (B); slow migrating fragments represent telomere end-to-end fusions and signal in native condition represents the single-stranded telomeric G-overhang. Mean and s.d. scored from triplicate experiments; *P < 0.05 as determined by two-tailed Student’s t-test. D Localization of 53BP1 to telomeric DNA (TTAGGG). E Graph representing the percentage of cells with telomere dysfunction-induced foci (TIFs); Mean and s.d. scored from triplicate experiments, n > 200; *P < 0.05 as determined by two-tailed Student’s t-test. F FACS mediated cell cycle analysis of the indicated cells. G ATM activation following TRF2 depletion as determined by Chk2 phosphorylation.

Figure 4. DNA binding activity of RNF4 is required for the DNA damage response

A Expression levels of MYC tagged RNF4 (WT) and RNF4 mutants [K179D (KD), SIM mutant (SIM)]. Tubulin (tub) was used as a loading control. B TRF2 null cells depleted for endogenous RNF4 (shRNF4) were complemented with the indicated constructs and stained for γH2AX and 53BP1. The graph represents the proportion of cells with γH2AX and 53BP1 containing foci relative to control cells (shLuc). Mean and s.d. scored from triplicate experiments, n > 200; *P < 0.05, calculated using a two-tailed Student’s t-test. C Metaphases harvested from cells treated as indicated were stained for telomeric DNA (green) and DAPI (red). Percentages of fused chromosome ends are indicated.

Figure 5. RNF4 acts downstream of ATM kinase signaling

A Mouse embryonic fibroblasts (MEFs) infected with the indicated constructs were stained for γH2AX and 53BP1. B MEFs infected with the indicated constructs were irradiated with 2 Gy and stained for 53BP1 and γH2AX. C The graph represents the percentage of cells that display > 15 DNA damage foci containing 53BP1 and γH2AX. Mean and s.d. scored from triplicate experiments, n > 200. D Quantitation of cells containing > 20 53BP1 foci at the indicated time post-ionizing radiation exposure. Mean and s.d. scored from triplicate experiments; n > 200; *P < 0.05, calculated using a two-tailed Student’s t-test. E Quantitation of 53BP1 foci 10 min after irradiation (2 Gy) in MEFs expressing the indicated shRNA and RNF4 WT, K179D (KD), SIM mutant or empty vector (EV). Foci are normalized to mock treated cells with EV. Mean and s.d. scored from triplicate experiments; n > 200; *P < 0.05, calculated using a two-tailed Students t-test.