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. 2014 Jul;42(12):7720-33.
doi: 10.1093/nar/gku421. Epub 2014 May 14.

An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells

Meilen C Muñoz  1 , Diana A Yanez  2 , Jeremy M Stark  3
Affiliations

An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells

Meilen C Muñoz et al. Nucleic Acids Res. 2014 Jul.

Abstract

The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.

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Figures

Figure 1.
Figure 1.
Induction of chromosomal breaks by distinct nucleases to examine HR and EJ. (A) Shown are diagrams of the DR-GFP and EJ5-GFP reporters for measuring HDR and Distal-EJ, respectively. Also shown are guide RNA (gRNA) targeting sequences used for inducing chromosomal breaks with the gRNA/CAS9 system (see the lines below the italicized sequence labeled gDR, gEJ5-5 and gEJ5-3), with the predicted cleavage site denoted by a triangle. Since the gRNAs include sequences flanking the I-SceI recognition site, each gRNA targets a unique site on the reporters. The I-SceI recognition site is in lower case. (B) Repair events induced by I-SceI, CAS9-WT and CAS9-D10A. The expression cassettes for the gRNAs and CAS9 are present on the same plasmid. U2OS cells with the DR-GFP and EJ5-GFP reporters were transfected with a set of gRNA/CAS9 plasmids, as well as I-SceI, or left untransfected. Shown are representative flow cytometry plots (FL1/green on the Y-axis, FL2 on the X-axis) from these transfections, with the GFP+ population delineated in the upper gate, used to determine the mean frequencies of GFP+ cells (n = 3). (C) CAS9-WT is proficient at inducing loss of the I-SceI site via mutagenic Proximal-EJ, which is further increased with co-expression of the exonuclease Trex2. U2OS cells with the EJ5-GFP reporter were transfected with I-SceI and CAS9-WT as in B, except including either the Trex2 expression vector or empty vector (EV). Shown are representative amplification products from these transfections digested with the I-SceI endonuclease, which were used to determine the frequency of I-SceI site loss (n = 6, P < 0.03). Also shown is the frequency of Distal-EJ from the transfections of CAS9-WT with or without Trex2 (*P < 0.0001).
Figure 2.
Figure 2.
An N-terminal fragment of RNF168 (N221*) is proficient at inhibiting HR in BRCA1 deficient cells, in a manner dependent on the charge of R57. (A) Depletion of RNF168 and BRCA1 via siRNA and expression of RNF168 mutant forms. Shown is a diagram of RNF168 with approximate positions of the R57D and N221* mutations, as well as the RING, MIU1 and MIU2 motifs. Immunoblot signals are shown for BRCA1, RNF168 and actin for U2OS cells treated with siRNAs targeting BRCA1 and RNF168 (siBRCA1#6, siRNF168#18), and a non-targeting siRNA (siCTRL). Subsequent to siRNA treatment, cells were transfected with expression vectors for Flag-tagged RNF168 (WT, R57D, N221*, R57D/N221*, each resistant to siRNF168#18) or EV. Shown are immunoblot signals from these transfections for RNF168 (WT and R57D), Flag (N221* and R57D/N221*) and actin. (B) Analysis of RNF168 mutant forms for inhibition of I-SceI-induced HR in BRCA1 depleted cells. Two U2OS reporter cell lines that measure distinct types of HR were evaluated: DR-GFP that measures HDR and SA-GFP that measures SSA. These U2OS cell lines were treated with the siRNAs described in A and subsequently co-transfected with the expression vector for I-SceI along with the RNF168 expression vectors described in A, or the control EV. Shown are the frequencies of GFP+ cells for each reporter cell line, relative to parallel transfections with a non-targeting siRNA (siCTRL) and control EV. *P < 0.0001 (n = 6). (C) RNF168 influences HDR of CAS9-induced chromosomal breaks similarly to those induced by I-SceI. U2OS DR-GFP reporter cell line was transfected as in B, except replacing I-SceI with gRNA/CAS9 plasmids expressing gDR and CAS9-WT or CAS9-D10A, as shown in Figure 1. Frequencies of GFP+ cells were measured as in B. *P ≤ 0.022 (n = 6).
Figure 3.
Figure 3.
RNF168-N221* is deficient at forming IRIF, but proficient at promoting 53BP1 IRIF and ubiquitination of H2AX at K13/15, in a manner dependent on the charge of R57. (A) Analysis of RNF168 mutants for proficiency at forming IRIF and promoting 53BP1 IRIF. U2OS cells were treated with siCTRL or siBRCA1 + siRNF168 and subsequently transfected with each of the Flag-tagged RNF168 expression vectors described in Figure 2A. Subsequently, cells were treated with 6 Gy of IR (Cs137), and allowed to recover 4 h prior to fixation and co-immunostaining with Flag and 53BP1 antibodies. Shown are Flag and 53BP1 immunostaining, and DAPI staining for representative cells from each transfection. Scale bar = 10 μm. (B) Expression of RNF168-N221* promotes H2AX ubiquitination at K13/15 in a manner dependent on the charge of R57. Mouse H2ax−/− embryonic stem cells were transfected with expression vectors for H2AX with various lysine to glutamine mutations at previously described ubiquitination sites: two double mutants (K13/15Q, K118/119Q) and one quadruple mutant (K13/15/118/119Q). We analyzed each of these mutants because K118/119 monoubiquitination is predominant; such that K13/15 monoubiquitination is only detectable in the K118/119Q mutant. Cells were also co-transfected with expression vectors for RNF168, subsequently treated with 10 Gy IR (Cs137), and allowed to recover for 2.5 h prior to both soluble protein and histone extraction in the presence of a de-ubiquitination inhibitor. Shown are immunoblotting signals for H2AX from the histone extract of these samples: *denotes a non-specific band observed in untransfected cells and Ub1 denotes migration of H2AX at a position consistent with monoubiquitination. Also shown are Flag and actin immunoblotting signals for cells expressing Flag-tagged RNF168-N221 and R57D/N221*.
Figure 4.
Figure 4.
RNF8 depletion causes a modest increase in HR in BRCA1 deficient cells that can be reversed by expression of RNF168 WT and N221*. (A) Depletion of RNF8 and BRCA1 via siRNA and expression of RNF8. U2OS cells treated with siRNAs targeting BRCA1 and RNF8 (siBRCA1#7, siRNF8#5), and a non-targeting siRNA (siCTRL). Subsequent to siRNA treatment, cells were transfected with an expression vector for RNF8 (siRNF8#5-resistant), RNF168, or EV. Shown are immunoblot signals from transfections for RNF8, BRCA1, RNF168, and actin. (B) Analysis of HDR induced by I-SceI for cells depleted of BRCA1 and RNF8. The U2OS DR-GFP reporter cell line was treated with the siRNAs described in A, and subsequently co-transfected with the expression vector for I-SceI. In addition, using samples treated with siRNAs targeting BRCA1 and RNF8, each of the RNF168 expression vectors described in Figure 2, as well as RNF8, were included in the I-SceI transfection. Shown are the frequencies of GFP+ cells for each reporter cell line, relative to parallel transfections with a non-targeting siRNA (siCTRL) and control EV. *P < 0.0001 (n = 6). (C) Analysis of HDR induced by CAS9 for cells depleted of BRCA1 and RNF8. Transfections were performed as in B, except replacing I-SceI with gRNA/CAS9 plasmids expressing gDR with CAS9-WT or CAS9-D10A, as shown in Figure 1. Frequencies of GFP+ cells were measured as in B. *P ≤ 0.034 (n = 3).
Figure 5.
Figure 5.
53BP1 IRIF can be restored in RNF8 depleted cells by expression of RNF168 WT and N221*. U2OS cells were treated with siCTRL or siBRCA1 and siRNF8 and subsequently transfected with a set of the Flag-tagged RNF168 expression vectors or the RNF8 expression vector, which are described in Figures 2A and 4A. Subsequently, cells were treated with 6 Gy of IR (Cs137), and allowed to recover for 4 h prior to fixation and immunostaining. RNF8 and 53BP1 antibodies were used for the RNF8 expression vector samples, and Flag and 53BP1 antibodies were used for the RNF168 expression vector samples. Shown are immunostaining, and DAPI staining images for representative cells from each transfection. Scale bar = 10 μm.
Figure 6.
Figure 6.
The LRM1 and ubiquitin binding motifs within RNF168-N221* are important but not essential for inhibition of HR. (A) Conserved motifs within N221* are important for inhibition of HDR. Shown is a diagram of N221* with the approximate positions of the LRM1 and UMI/MIU1 motifs. Also shown are immunoblotting signals for Flag and actin for cells transfected with a set of expression plasmids for Flag-RNF168-N221* with mutations in these conserved motifs, as well as Flag-RNF168-N221-571. U2OS DR-GFP cells were treated with siRNAs targeting BRCA1 and RNF168, and subsequently co-transfected with expression vectors for I-SceI and a set of RNF168 mutants. Shown are the frequencies of GFP+ cells for each reporter cell line, relative to parallel transfections with a non-targeting siRNA (siCTRL) and control EV. *distinct from EV, P < 0.0001 (n = 6). (B) RNF168 mutants that show at least intermediate inhibition of HR are also proficient for promoting 53BP1 IRIF. U2OS cells were treated with siCTRL or siBRCA1 and siRNF168 and subsequently transfected with Flag-tagged RNF168 expression vectors described in A. Subsequently, cells were treated with 6 Gy of IR (Cs137), and allowed to recover for 4 h prior to fixation and immunostaining. Shown are Flag and 53BP1 immunostaining, and DAPI staining images for representative cells from each transfection. The exposure times for each type of immunostain are the same for each cell. The cells were selected to represent the 53BP1 IRIF results for each RNF168 mutant as quantified in Table 2, but do not necessarily represent the average intensity of Flag staining for each RNF168 mutant, which can show variability among cells. Scale bar = 10 μm.
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