1. Volume 50, Issue 3, Pages 333-343 (May 2013)
Prolyl Isomerase PIN1 Regulates DNA Double-Strand Break Repair by Counteracting DNA End Resection 
Martin Steger, Olga Murina, Daniela Hühn, Lorenza P. Ferretti, Reto Walser, Kay Hänggi, Lorenzo Lafranchi, Christine Neugebauer, Shreya Paliwal, Pavel Janscak, Bertran Gerrits, Giannino Del Sal, Oliver Zerbe, Alessandro A. Sartori 
Molecular Cell 
Volume 50, Issue 3, Pages (May 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2013 50, 333-343DOI: (10.1016/j.molcel.2013.03.023)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 PIN1 Regulates DSB Repair
(A) HEK293 EJ5-GFP cells were transfected with the indicated siRNAs. Two days later, cells were transfected with the I-SceI expression plasmid and harvested after 48 hr for flow cytometry and immunoblot analysis.
(B) HEK293 DR-GFP cells were transfected with the indicated siRNAs and further processed as in (A).
(C) HEK293 DR-GFP cells were cotransfected with the indicated HA-PIN1 variants together with the I-SceI plasmid and further processed as in (A).
(D) Control- or PIN1-depleted U2OS cells were treated with etoposide (ETOP, 20 μM) for 6 hr, and whole-cell extracts were analyzed by immunoblotting. Asterisks indicate hyperphosphorylated forms of CtIP and RPA2, respectively.
(E) Pin1−/− MEFs complemented with empty vector (EV, pLPC) or PIN1 were treated with ETOP (10 μM) for 2 hr and lysed at the indicated times after ETOP removal. Whole-cell extracts were analyzed by immunoblotting, and the amount of broken DNA was assessed by PFGE followed by ethidium bromide (EtBr) staining (see also Figure S2F for quantification of the PFGE signals).
(F) HEK293T cells were transfected with empty vector (EV, pcDNA3.1) or HA-PIN1-wt for 72 hr. Cells were irradiated (30 Gy) and whole-cell extracts were prepared at indicated times and analyzed by immunoblotting. Asterisk indicates hyperphosphorylated form of RPA2.
(G) Forty-eight hours after transfection with the indicated siRNAs, U2OS cells grown on coverslips were treated with ETOP (5 μM) for 1 hr, fixed, and coimmunostained for γ-H2AX and RPA2 (see also Figure S2H). In each sample at least 50 cells were scored. Graph shows the percentage of cells exhibiting more than 10 RPA foci/nuclei. Immunoblot analysis of the same samples is shown below.
(H) Shown is NHEJ assay and immunoblot analysis after transfection with the indicated siRNAs as in (A). In (A), (B), (C), (G), and (H), data are represented as mean ± SEM (n ≥ 3). See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 2 PIN1 Interacts with CtIP through Phosphorylated S/T-P Motifs
(A) GST-PIN1-wt or -W34A fusion proteins were immobilized on glutathione-Sepharose beads and incubated with whole-cell extracts from either U2OS cells (lanes 1–3) or U2OS cells stably expressing GFP-CtIP (lanes 4–6). Inputs and precipitated bead fractions from the pull-downs were subjected to immunoblotting with anti-CtIP antibodies.
(B) (Left) Detection of endogenous PIN1-CtIP complexes by in situ proximity ligation assay (PLA). U2OS cells were pulse labeled with 5′-ethynyl-2′-deoxyuridine (EdU, 10 μM) for 15 min, fixed, and incubated with antibodies against PIN1 and CtIP prior to detection of protein-protein interactions using a fluorescently labeled probe (PLA-613). Nuclei were visualized by DAPI staining, and EdU incorporation was detected according to the manufacturer’s instructions (see also Figure S3C). (Right) Quantification of the PLA signals/cell. For both conditions, PLA signals from at least 50 cells were enumerated. Scale bar, 5 μm.
(C) (Left) GST-PIN1 pull-down assay using extracts of HEK293T cells expressing indicated FLAG-tagged versions of CtIP. The band intensities were quantified using ImageJ and represented as input/pull-down (I/P) ratios. (Right) Data are represented as mean values of densitometric quantification ± SEM (n ≥ 5).
(D) HEK293T cells were cotransfected with HA-PIN1 and the indicated FLAG-CtIP plasmids. HA-PIN1 was immunoprecipitated from whole-cell extracts using anti-HA antibody, and immunocomplexes were analyzed by western blotting.
(E) Anti-FLAG immunoprecipitates from empty vector- or FLAG-CtIP-transfected HEK293T cells were subjected to far-western blotting using purified GST-PIN1 as a probe, followed by immunodetection with anti-GST antibody. After stripping, the same membrane was reprobed using anti-FLAG antibody.
(F) HEK293T cells were cotransfected with GFP-CtIP (wt and 2A) and HA-PIN1 for 48 hr before treatment with ETOP (10 μM) for 2 hr. Whole-cell extracts were analyzed by western blotting before (input) and after immunoprecipitation (IP) using anti-GFP antibody. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3
CtIP-T315 and CtIP-S276 Are Phosphorylated to Promote PIN1 Binding and cis/trans Isomerization
(A) Extracts from HEK293T cells transfected for 48 hr with the indicated FLAG-CtIP constructs were immunoblotted with either rabbit polyclonal antibodies raised against CtIP phosphopeptides or with anti-FLAG antibody.
(B) Extracts from HEK293T cells were treated with λ-PPase, immunoprecipitated using anti-CtIP antibody, and immunoblotted with the indicated antibodies.
(C) GST-PIN1 pull-down assays were performed using U2OS whole-cell extracts (0.5 mg) supplemented with the indicated CtIP peptides (80 μg).
(D) Shown is selected region of the two-dimensional ROESY spectra of the CtIP-pS276 peptide after incubation with purified, recombinant GST-PIN1.
(E) HEK293T cells were transfected with the indicated FLAG-CtIP constructs for 72 hr. CtIP proteins were purified using M2 magnetic beads, eluted with 3xFLAG peptides, digested with V8 protease, and analyzed by western blotting using anti-CtIP antibody. See also Figure S4 and Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 4
CtIP-PIN1 Interaction and CtIP-T315 Phosphorylation Require CDK2 Activity
(A) Extracts from U2OS cells treated for 2 hr with DMSO, R-Roscovitine (25 μM), or RO-3306 (25 μM) were subjected to GST-PIN1 pull-down assays.
(B) HEK293T cells were treated for 2 hr with DMSO or ROSC, and extracts were immunoblotted for CtIP before (input) and after immunoprecipitation (IP) using the anti-CtIP-pT315 antibody.
(C) HEK293 cells expressing GFP-CtIP (wt and 2A) were treated for 3 hr with either DMSO or ROSC (25 μM). After lysis, whole-cell extracts were immunoblotted for GFP either directly (input) or after immunoprecipitation (IP) with anti-pT315 or anti-pS276 antibodies.
(D) Extracts from HEK293T cells transfected with either pcDNA3.1 (−) or plasmids expressing HA-tagged dominant-negative (dn) mutants of CDK1, CDK2, and CDK4 for 48 hr were subjected to GST-PIN1 pull-down assays. The ratios of input versus pull-down (I/P; in A and D) and input versus IP (I/IP; in B and C) were quantified by densitometry using ImageJ. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

7. Figure 5 CtIP-2A Mutant Promotes Hyperresection of DSBs
(A) U2OS cells stably expressing siRNA-resistant GFP-tagged CtIP-wt and CtIP-2A or the empty vector (−) were transfected with CNTL or CtIP siRNA for 72 hr, and whole-cell extracts were analyzed by western blotting.
(B) The same cells as in (A) were analyzed by flow cytometry.
(C) The same cells as in (A) were sensitized with BrdU followed by laser microirradiation. After 30 min, cells were fixed, coimmunostained for γ-H2AX and cyclin A, and analyzed by fluorescence microscopy (see also Figure S7B).
(D) The same cells as in (A), including the resection-defective CtIP-T847A mutant, were transfected with CtIP siRNA for 72 hr and treated for 1 hr with either DMSO or low doses of CPT (see also Figure S7C). Survival was determined by colony formation. Data are represented as mean ± SEM (n = 3).
(E) The same cells as in (A) were treated with ETOP (5 μM) for 1 hr, released into drug-free medium for the indicated times, and analyzed by immunoblotting.
(F) The same cells as in (A) were treated with ETOP (5 μM) for 1 hr and were either immediately fixed or released into drug-free medium for 2 hr before fixation. After pre-extraction, cells were coimmunostained for RPA2 and γ-H2AX and analyzed by fluorescence microscopy (see also Figure S7D). For each condition at least 50 cells were scored. Graph shows the percentage of cells exhibiting more than 10 RPA foci per nuclei. Data are represented as mean ± SEM (n ≥ 2).
(G) The same cells as in (A) were treated with ETOP (10 μM) for 2 hr in the absence or presence of a DNA-PKcs inhibitor (NU7441, 10 μM). Cells were harvested either directly or at 30 min after the release into drug-free medium. Whole-cell extracts were analyzed by immunoblotting, and genomic DNA was analyzed by PFGE. DNA breakage in each lane was quantified using ImageJ and normalized against intact DNA. Relative amount of broken DNA in cells expressing GFP-CtIP-wt treated with ETOP was set to 100%.
(H) Same cells as in (A) were synchronized by a single thymidine block. Eight hours after the release from thymidine, cells enriched in S/G2 were irradiated at 30 Gy and, 2 hr later lysates were analyzed by immunoblotting. In (E), (G), and (H), the asterisk indicates the hyperphosphorylated form of RPA2. See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

8. Figure 6 PIN1 Destabilizes CtIP by Promoting Its Ubiquitylation
(A) Two days after transfection with the indicated plasmids, HEK293 cells were treated with CPT (1 μM) for 2 hr and whole-cell extracts were analyzed by immunoblotting. Asterisk indicates hyperphosphorylated form of CtIP.
(B) HEK293T cells were transfected with either FLAG-CtIP-wt or FLAG-CtIP-2A. Eight hours after transfection, cells were splitted into two new plates. Twenty-four hours after plasmid transfection, cells were treated with MG132 (10 μM) for 8 hr and lysed for immunoblot analysis using the indicated antibodies. The signal intensities of CtIP bands were quantified by densitometric analysis using the ImageJ software and normalized to those of Actin. The values represent the relative increase in CtIP-wt and CtIP-2A levels upon MG132 treatment.
(C) Three days after transfection with CtIP siRNA, U2OS cells stably expressing siRNA-resistant GFP-CtIP (wt and 2A) were treated with DMSO or ETOP (10 μM). After 1 hr, cells were released into fresh medium supplemented with 200 μg/ml cycloheximide (CHX) or not for the indicated times, and lysates were analyzed by immunoblotting (see also Figure S8).
(D) HEK293/Flp-In/T-REx cells containing stably integrated GFP-CtIP constructs (wt and 2A) were cultivated in the absence or presence of doxycycline (Dox; 1 μg/ml) for 24 hr, and lysates were analyzed by immunoblotting.
(E) The same cells as in (D) were treated with Dox for 24 hr. After lysis, whole-cell extracts were analyzed by western blotting either directly (input) or after immunoprecipitation (IP) with the indicated anti-CtIP phospho-specific antibodies.
(F) Forty-eight hours after transfection with the indicated siRNAs, HEK293/Flp-In/T-REx cells were transfected with His-Ub, and the expression of GFP-CtIP-wt was simultaneously induced with Dox (except in lane 1). Eight hours after induction, cells were transfected with siRNA for a second time. Seventy-two hours after the first siRNA transfection, cells were treated with MG132 (20 μM) for 6 hr, followed by lysis in buffer containing guanidium-HCl. Ubiquitin conjugates were purified using Ni-NTA-agarose beads, eluted, and analyzed by western blotting using anti-GFP antibody.
(G) Thirty hours after transfection with His-Ub, Dox-induced HEK293/Flp-In/T-REx cells expressing either GFP-CtIP-wt or GFP-CtIP-2A were lysed in buffer containing guanidium-HCl and processed as in (F).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

9. Figure 7
Hypothetical Model: How PIN1-Mediated CtIP Isomerization Controls DNA End Resection
During S/G2, CtIP together with other nucleases promotes the resection of DSBs. Following resection initiation, proline-directed kinases including CDK2 phosphorylate CtIP on T315 and S276, resulting in the binding of PIN1 to CtIP. PIN1-mediated isomerization of CtIP leads to CtIP ubiquitylation through an as-yet-unknown E3 ubiquitin ligase and subsequent CtIP degradation by the proteasome. This mechanism ensures an appropriate usage of DSB end resection. Consequently, cells with abrogated PIN1 function or inherently low PIN1 levels display reduced NHEJ and aberrant (error-prone) forms of homology-directed repair due to enhanced CtIP resection activity (hyperresection). In contrast, cells overexpressing PIN1 display reduced HR and increased NHEJ due to decreased CtIP resection activity (hyporesection). Therefore, we propose that PIN1 plays an important role in the regulation of DSB repair, particularly in late S and G2 phases of the cell cycle.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions