A PALB2-interacting domain in RNF168 couples homologous recombination to DNA break-induced chromatin ubiquitylation
- Leiden University Medical Center, The Netherlands
- CHU de Québec Research Center, HDQ Pavilion, Oncology Axis, McMahon, Canada
- Laval University Cancer Research Center, Canada
Figures
Figure 1 with 1 supplement
RNF168-dependent recruitment of PALB2 to chromatin.
(A) Schematic of the tethering system. (B) PALB2 (white) accumulation upon tethering of the indicated mCherry-LacR fusion proteins (red) in cells containing a LacO array. (C) Quantification of B. (D)…
Figure 1—figure supplement 1
RNF168-dependent recruitment of 53BP1 to chromatin.
(A) Conjugated ubiquitin (FK2; white) and 53BP1 (white) accumulation upon tethering of the indicated mCherry-LacR fusion proteins (red) in cells containing a LacO array. (B) Quantification of A. (C) …
Figure 2 with 1 supplement
RNF168 promotes PALB2 and RAD51 recruitment and HR.
(A) Effect of the indicated siRNAs on PALB2 (white) IRIF formation at 6 hr after 10 Gy in mAG-geminin-expressing (green) S/G2 U2OS cells. (B) RAD51 (white) IRIF formation at 6 hr after 10 Gy in …
Figure 2—figure supplement 1
RNF168 promotes RAD51 recruitment and is epistatic with PALB2 in HR.
(A) BRCA1 or RAP80 accumulation in control (siLuc) cells or in cells depleted for either BRCA1 or RAP80 to validate the knock-down of these genes in Figure 2D. (B) Quantification of A. (C) RAD51 …
Figure 3 with 1 supplement
RNF168 does not affect recruitment of BRCA1 to HR sites in S/G2 cells.
(A) BRCA1 (green) and 53BP1 (white) IRIF formation in mCherry-geminin-expressing (red) S/G2 U2OS cells transfected with siLuc. (B) As in A, except that cells were transfected with siRNF168. (C) …
Figure 3—figure supplement 1
Model for the spreading of proteins from DSBs.
Schematic representation of the spreading of HR (orange) and signaling (green) proteins from a DSB, resulting in different foci sizes after IR as measured in Figure 3.
Figure 4 with 1 supplement
PALB2 directly interacts with RNF168 via its WD40 domain.
(A) Pulldowns of the indicated GFP fusion proteins in U2OS cells. Blots were probed for PALB2 and GFP. (B) In vitro GST pulldown of GST-PALB2 or GST alone in the presence of His-RNF168. Blots were …
Figure 4—figure supplement 1
RNF168 and BRCA2 interact with PALB2 in a non-mutually exclusive manner.
(A) Pulldowns of the indicated GFP and YFP fusion proteins in U2OS cells. Blots were probed for PALB2, BRCA2 and GFP. (B) In vitro GST pulldown of GST-PALB2 or GST alone in the presence of …
Figure 5
RNF168-dependent recruitment of PALB2 requires its WD40 domain.
(A) Schematic representation of full-length PALB2 (WT) and the WD40 deletion mutant (T7). (B) Schematic of the tethering system. (C) Recruitment of the indicated YFP-PALB2 variants (green) upon …
Figure 6 with 1 supplement
RNF168-mediated PALB2 recruitment is ubiquitylation dependent.
(A) Recruitment of ubiquitin conjugates (FK2; white) and endogenous PALB2 (green) upon tethering of the indicated mCherry-LacR-RNF168 variants (red) to a genomic LacO array in U2OS 2-6-3 cells. (B) …
Figure 6—figure supplement 1
PALB2 neither associates with ubiquitin, nor stimulates the association of RNF168 with ubiquitin in vitro.
(A) Schematic representation of full-length PALB2 (WT) and five non-overlapping fragments (T1–T5) spanning PALB2. (B) Pulldowns of GST alone or the indicated GST fusion proteins in the presence of …
Figure 7 with 3 supplements
PALB2 associates with K63-polyubiquitin chains via RNF168.
(A) GST pulldowns to assess the binding of GST-PALB2 or GST-alone to K63 polyubiquitin (pUb) chains in the presence or absence of His-RNF168. Blots were probed for GST, ubiquitin and His. (B) …
Figure 7—figure supplement 1
RNF168 RING mutants do not support IR-induced 53BP1 focus formation.
(A) 53BP1 IRIF formation (orange) at 1 hr after 2 Gy in γH2AX-positive cells (white) transfected with the indicated siRNAs and siRNA-resistant GFP-tagged RN168 cDNAs (green) to validate the mutants …
Figure 7—figure supplement 2
Expression of H2A lacking K13 and K15 decreases 53BP1 recruitment to FokI-induced DSBs.
(A) Recruitment of 53BP1 (green) to DSBs induced by FokI-mCherry-LacR at a LacO array (red) in U2OS 2-6-5 cells expressing the indicated FLAG-H2A constructs (white) to validate the experimental …
Figure 7—figure supplement 3
Model for RNF168’s role in PALB2 recruitment.
Schematic representation of the RING-dependent ubiquitylation of H2A by RNF168, which is followed by the MIU-dependent binding of RNF168 to ubiquitylated H2A. The direct protein-protein interaction …
Figure 8 with 3 supplements
Recruitment of PALB2 requires its interaction with RNF168’s PID.
(A) Schematic representation of full-length RNF168 (WT) and five deletion mutants (T1–T5). (B, C) Pulldowns of the indicated GFP fusion proteins in U2OS cells. Blots were probed for PALB2 and GFP. (D…
Figure 8—figure supplement 1
Conservation of the RNF168 PID in mammals.
Alignment of the PID (amino acids 524–571) from human RNF168 with the C-terminus of RNF168 orthologues from other placental mammals generated using Clustal Omega software (http://www.ebi.ac.uk/Tools/…
Figure 8—figure supplement 2
RNF168 PID mutants fully support IR-induced 53BP1 focus formation.
(A) Schematic representation of full-length RNF168 (RNF168WT) and the two C-terminal deletion mutants RNF168Δ479-571 (T4) and RNF168Δ525-571 (T5). (B) 53BP1 IRIF formation (orange) at 1 hr after 2 …
Figure 8—figure supplement 3
Stable inducible expression of GFP-tagged proteins in HeLa cells.
Expression levels of endogenous RNF168 and ectopic GFP-RNF168WT or GFP-RNF168Δ525-571 (T5) in stable HeLa Flp-In/T-REx cells as determined by Western blot (WB) analysis. To determine the optimal …
Additional files
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Supplementary file 1
siRNAs and antibodies used in this study.
(A) List of siRNAs and (B) List of antibodies.
- https://doi.org/10.7554/eLife.20922.021
