Journal: The Journal of Experimental Medicine

Article Title: Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

doi: 10.1084/jem.20191688

Figure Lengend Snippet: Knockdown of ZBTB24 does not affect cell cycle progression and the expression of genes involved in DSB repair. ICF2 patient-derived fibroblasts and ZBTB24 KO U2OS cells are not sensitive to IR (related to ). (A) U2OS cells and HEK293T cells containing the EJ5-GFP reporter were treated with the indicated siRNAs. WCEs were prepared 48 h later and subjected to Western blot analysis for ZBTB24. RAD51 is a loading control. (B) HEK293T cells containing the EJ5-GFP reporter were transfected with the indicated siRNAs. 48 h later, cells were transfected with a control vector or the I- Sce I expression vector (pCBASce). After an additional 24 h, cells were subjected to propidium iodide staining followed by flow cytometry analysis. The percentage of cells in G1 (red bar), S (blue bar), and G2/M (green bar) phase is presented. The mean ± SEM from two independent experiments is shown. Statistical significance was calculated using Student’s t test (ns, not significant). (C) Schematic of the GC92 reporter for NHEJ. (D) Fibroblasts containing the GC92 reporter were treated with the indicated siRNAs and, 48 h later, cotransfected with I- Sce I (pCBASce) and mCherry expression vectors. The ratio of CD4-FITC/mCherry-expressing cells was counted by flow cytometry 48 h later. The mean ± SEM from three to four independent experiments is shown. Statistical significance was calculated using Student’s t test (**, P < 0.01; ****, P < 0.0001). (E) Cells from D were subjected to Western blot analysis of KU80 and ZBTB24 expression. Tubulin and RAD51 are loading controls. (F) U2OS cells were treated with the indicated siRNAs. WCEs were prepared 48 h later and subjected to Western blot analysis for DNA-PKcs and PARP1. Tubulin is a loading control. (G) HEK293T cells were treated with control siRNAs against luciferase or three different siRNAs against ZBTB24. 4 d later, RNA was isolated and subjected to RNA sequencing analysis. The number of genes found to be commonly misregulated after ZBTB24-depletion with each of the siRNAs is presented (false discovery rate < 0.05). Importantly, gene ontology term term analysis (0006302; DSB repair) did not reveal the presence of DSB repair genes among the misregulated genes. (H) ICF2 patient-derived fibroblasts were exposed to different doses of IR and scored for clonogenic survival. The mean ± SEM from two independent experiments is shown. (I) ZBTB24 KO U2OS cells were exposed to different doses of IR and scored for clonogenic survival. The mean ± SEM from two independent experiments is shown. (J) Western blot analysis of ZBTB24 and CDCA7 expression in ZBTB24 KO U2OS clones from I. Tubulin is a loading control. (K) TIDE analysis of ZBTB24 KO clones from I , showing 17- and 13-bp out-of-frame deletions in ZBTB24 KO-1 and 11 and 10-bp out-of-frame deletions in ZBTB24 KO-2.

Article Snippet: Briefly, IP samples were resolved on 8% denaturing PAGE gels along with purified human PARP1 (Aparptosis) as a positive control.

Techniques: Knockdown, Expressing, Derivative Assay, Western Blot, Control, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Luciferase, Isolation, RNA Sequencing, Clone Assay

Journal: The Journal of Experimental Medicine

Article Title: Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

doi: 10.1084/jem.20191688

Figure Lengend Snippet: PARP1 interacts with ZBTB24 in a PARylation-dependent manner and recruits ZBTB24 to sites of DNA damage. (A) Schematic representation of SILAC-based MS approach. GFP- or GFP-ZBTB24–expressing U2OS cells were labeled with Lys0 and Arg0 (L) or Lys8 and Arg10 (H), respectively. Lysates were subjected to GFP IP, and equal amounts of both IP fractions were mixed. Proteins in the IP fractions were digested by trypsin and subjected to MS analysis. A list of ZBTB24-interacting proteins, including the number of peptides and the interaction ratio from heavy (H)- over light (L)-labeled cell extracts as revealed by MS, is shown. (B) As in A, but with GFP- and GFP-PARP1–expressing U2OS cells. (C) Cells expressing GFP-ZBTB24, GFP-PARP1, and Myc-ZBTB24 or GFP-NLS and Myc-ZBTB24 were treated with either DMSO (Mock) or PARP inhibitor (PARPi). Whole-cell extracts (WCEs) were subjected to GFP IP followed by Western blot analysis of the indicated proteins. (D) IP of endogenous PARP1 in U2OS cells. IgG is a negative control. Blots were probed for ZBTB24 and PARP1. (E) Schematic representation of the laser microirradiation approach. (F) GFP-ZBTB24 or ZBTB24-GFP accumulate at γH2AX-decorated DNA damage tracks after transient expression and laser microirradiation in U2OS cells. Scale bar, 10 µm. (G) As in F, except that cells transiently expressing GFP-ZBTB24 and mCherry-NBS1 were treated with either DMSO (Mock) or PARPi before GFP-ZBTB24 and mCherry-NBS1 accumulation was monitored at the indicated time points after laser microirradiation. Scale bar, 10 µm. (H) Quantification of the results from G. The mean ± SEM of two to three independent experiments is shown. (I) As in G, except that cells were cotransfected with GFP-ZBTB24 and the indicated siRNAs. Scale bar, 10 µm. (J) Quantification of the results from I. The mean ± SEM of two to three independent experiments is shown (left). Western blot showing the knockdown efficiency of PARP1 and PARP2 (right).

Article Snippet: Briefly, IP samples were resolved on 8% denaturing PAGE gels along with purified human PARP1 (Aparptosis) as a positive control.

Techniques: Multiplex sample analysis, Expressing, Labeling, Western Blot, Negative Control, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

doi: 10.1084/jem.20191688

Figure Lengend Snippet: PARG-dependent turnover of PAR chains modulates the accumulation of ZBTB24 at sites of DNA damage. The ZNF domain of ZBTB24 accumulates at sites of DNA damage in a PARP-dependent manner. ZBTB24 is not PARylated after DNA damage induction (related to and ). (A) U2OS cells transiently expressing GFP-ZBTB24 or GFP-NLS were treated with either DMSO (Mock) or PARPi. WCEs were subjected to GFP IP followed by Western blot analysis of the indicated proteins. (B) U2OS cells transfected with the indicated GFP-tagged proteins were treated with either DMSO (Mock) or PARPi. WCEs were prepared and subjected to Western blot analysis to assess total PAR levels. (C) Western blot analysis showing total PAR levels in U2OS cells transfected with the indicated siRNAs and transiently expressing GFP-ZBTB24. Tubulin is loading control. (D) GFP-ZBTB24 accumulation as monitored at the indicated time points after laser microirradiation in cells from C . Scale bar, 10 µm. (E) Quantification of the results from D. The mean ± SEM from two independent experiments is shown. (F) As in C , except that cells were cotransfected with a GFP-ZBTB24 and either an mCherry or mCherry-PARG expression vector were used. (G) As in D, except that cells from F were used. Scale bar, 10 µm. (H) Quantification of the results from G. The mean ± SEM from three independent experiments is shown. (I) U2OS cells transiently expressing GFP-tagged BAZ domains of ZBTB24 were treated with DMSO (Mock) or PARPi and subjected to laser microirradiation to follow GFP-BAZ accumulation at sites of DNA damage at the indicated time points after irradiation. Scale bar, 10 µm. (J) Quantification of I. The mean ± SEM from two independent experiments is shown. (K) As in I, except for the GFP-tagged ZNF domain of ZBTB24 (GFP-ZNF). Scale bar, 10 µm. (L) Quantification of K. The mean ± SEM from two independent experiments is shown. (M) U2OS cells expressing GFP were left untreated or treated with IR or MNNG. WCE were prepared and subjected to Western blot analysis for global PAR levels. (N) WCE extracts from M and from cells expressing GFP-ZBTB24 or GFP-PARP1 were subjected to GFP IP. Washes were performed under high-salt conditions to remove interacting proteins. Western blot analysis was done for the indicated proteins and PAR. The experiment was performed two times for PARP1 and four times for ZBTB24. Blots from a representative experiment are shown.

Article Snippet: Briefly, IP samples were resolved on 8% denaturing PAGE gels along with purified human PARP1 (Aparptosis) as a positive control.

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

doi: 10.1084/jem.20191688

Figure Lengend Snippet: The ZNF domain in ZBTB24 interacts with PAR and mediates its recruitment to sites of DNA damage. (A) Schematic representation of isoform 1 of ZBTB24 and its BTB-, DNA-binding AT hook- and 8 × C 2 H 2 ZNF domain. Protein domains were separated as indicated and fused to GFP for functional analysis. (B) Western blot analysis of WCEs from U2OS cells expressing the indicated GFP-tagged ZBTB24 domains. (C) Accumulation of the indicated GFP-tagged ZBTB24 domain in laser microirradiated U2OS cells. Representative images of unirradiated and irradiated cells (taken at the indicated time point after irradiation) are shown. Scale bar, 10 µm. (D) HEK293T cells expressing the indicated GFP-tagged ZBTB24 domains were subjected to GFP IP. GFP-purified proteins were resolved by gel electrophoresis, blotted onto a membrane, renatured, and incubated with radioactive PAR ( 32 P-PAR). Recombinant (rec.) PARP1 is a positive control. (E) Lysates from U2OS cells transiently expressing either GFP-NLS or the indicated GFP-tagged ZBTB24 domains were subjected to GFP IP and Western blot analysis for the indicated proteins.

Article Snippet: Briefly, IP samples were resolved on 8% denaturing PAGE gels along with purified human PARP1 (Aparptosis) as a positive control.

Techniques: Binding Assay, Functional Assay, Western Blot, Expressing, Irradiation, Purification, Nucleic Acid Electrophoresis, Membrane, Incubation, Recombinant, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

doi: 10.1084/jem.20191688

Figure Lengend Snippet: ZBTB24 stimulates PARP1-dependent PAR synthesis and protects PAR chain stability. (A) U2OS cells transfected with the indicated siRNAs were left untreated or exposed to IR. 5 min later, whole-cell extracts (WCEs) were prepared and subjected to Western blot analysis for DNA-PKcs and PAR. DNA-PKcs is a loading control. (B) Quantification of the results from A and a second independent experiment. The mean ± SEM is shown. The ratio of PAR/loading control signals per sample was normalized to that of the IR-exposed siLuc sample, which was set to 1. Statistical significance was calculated using Student’s t test (*, P < 0.05; **, P < 0.01). (C) Schematic of the PAR synthesis assay. (D) Recombinant PARP1 was incubated with a damaged DNA template and activated by NAD + in the presence of increasing concentrations of GST-ZBTB24, GST-ZBTB24 ΔZNF, or GST only. The presence of 10H-PAR chains and recombinant proteins was monitored by Western blot analysis. (E) Quantification of ZBTB24-dependent stimulation of PAR synthesis from D and two other independent experiments. The mean ± SD is shown. The signal of 10H-PAR for each sample containing GST-ZBTB24 or GST-ZBTB24 ΔZNF was normalized to that without GST-ZBTB24, which was set to 1. Statistical significance was calculated using Student’s t test (*, P < 0.05). (F) Schematic of the PAR protection assay. (G) Recombinant PARP1 was incubated with a damaged DNA template and activated by NAD + to generate PARylated PARP1. Increasing concentrations of GST-ZBTB24, GST-ZBTB24 ΔZNF, or GST alone were added, followed by incubation with PARG. The presence of 10H-PAR chains and recombinant proteins was monitored by Western blot analysis. (H) As in E, except that PAR protection was measured from G and another independent experiment. The mean ± SD is shown. Statistical significance was calculated using Student’s t test (*, P < 0.05).

Article Snippet: Briefly, IP samples were resolved on 8% denaturing PAGE gels along with purified human PARP1 (Aparptosis) as a positive control.

Techniques: Transfection, Western Blot, Control, Recombinant, Incubation

Journal: The Journal of Experimental Medicine

Article Title: Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

doi: 10.1084/jem.20191688

Figure Lengend Snippet: Purification of recombinant ZBTB24 and PARP1 promotes XRCC4/LIG4 assembly and NHEJ at DNA damage sites. Related to and . (A) Coomassie-stained gel of recombinant GST, GST-tagged ZBTB24, and GST-tagged ZBTB24 ΔZNF, which were purified after expression in E. coli . The indicated samples from the purification procedure were loaded and run on a 4–12% polyacrylamide gel. (B) U2OS stably expressing GFP-XRCC4 were treated with DMSO (Mock) or PARPi and subjected to laser microirradiation. Representative images of unirradiated and irradiated cells (taken at the indicated time point after irradiation) are shown. Scale bar, 10 µm. (C) Quantification of B. The mean ± SEM from two independent experiments is shown. Statistical significance was calculated using Student’s t test (*, P < 0.05). (D) As in B, except that cells were transfected with the indicated siRNAs. Scale bar, 10 µm. (E) Quantification of D. The mean ± SEM from three to five independent experiments is shown. Statistical significance was calculated using Student’s t test (***, P < 0.001; ****, P < 0.0001). (F) Plasmid integration assays in U2OS cells transfected with indicated siRNAs. The mean ± SEM from two to four independent experiments is shown. Statistical significance was calculated using Student’s t test (**, P < 0.01; ****, P < 0.0001). (G) Accumulation of γH2AX and endogenous XRCC4 at sites of laser-inflicted DNA damage. U2OS cells were treated with the indicated siRNAs, subjected to laser microirradiation, and 10 min later, fixed and immunostained for γH2AX and endogenous XRCC4. Scale bar, 10 µm. (H) Quantification of endogenous XRCC4 levels in laser tracks from G. The mean ± SEM from two independent experiments is shown. Statistical significance was calculated using Student’s t test (****, P < 0.0001). (I) As in H, except for γH2AX. The mean ± SEM from two independent experiments is shown. Statistical significance was calculated using Student’s t test (ns, not significant).

Article Snippet: Briefly, IP samples were resolved on 8% denaturing PAGE gels along with purified human PARP1 (Aparptosis) as a positive control.

Techniques: Purification, Recombinant, Staining, Expressing, Stable Transfection, Irradiation, Transfection, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

doi: 10.1084/jem.20191688

Figure Lengend Snippet: ZBTB24-defiecent cells show a-NHEJ signatures at repair junctions. (A–C) Mutational signatures (A), deletion sizes (B), and microhomology usage (in case of deletion formation; C) at repair junctions in the GC92 reporter for NHEJ. GC92 cells were transfected with the indicated siRNAs and I- Sce I expression vector. Repair junctions were amplified by PCR and Sanger sequenced. The bars represent data obtained from three independent experiments. (D) Western blot analysis of Zbtb24 and AID expression in wild-type CH12 cells (Zbtb24 +/+ ), Zbtb24 +/− , and Zbtb24 −/− clones stimulated to undergo CSR for 3 d with TGF-β, IL-4, and an anti-CD40 antibody. β-Actin is a loading control. (E) Flow cytometry analysis of cells from D. The percentage of IgA-expressing cells is indicated. Representative contour plots of three independent experiments are shown. (F) Quantification of cells from E. The mean ± SD from three independent experiments is shown. Data were normalized to wild-type Zbtb24 +/+ cells, which were set to 100%. Statistical significance was calculated using Student’s t test (***, P < 0.001; ****, P < 0.0001; ns, not significant). (G) Western blot analysis of Zbtb24 and AID expression in Zbtb24 −/− clones transduced with an empty retrovirus (pMX-PIE) or a retrovirus expressing mZbtbt24 and EGFP cDNA (pMX-mZtbtb24) and stimulated to undergo CSR for 3 d with TGF-β, IL-4, and an anti-CD40 antibody. β-Actin is a loading control. (H) Flow cytometry analysis of cells from G. Dot plots are gated on EGFP + cells. The percentage of IgA-expressing cells is indicated. Representative contour plots of two independent experiments are shown. (I) Quantification of cells from H. The mean ± SD from two independent experiments is shown. Data were normalized to uninfected wild-type Zbtb24 +/+ cells, which were set to 100%. –, not infected. Statistical significance was calculated using one-way ANOVA (*, P < 0.05; ***, P < 0.001). (J) Model for the role of ZBTB24 in DSB repair by NHEJ. ZBTB24 accumulates at DSBs, where it functions as a scaffold to protect PARP1-associated PAR-chains, which serve as a docking site for the LIG4-XRCC4 complex, facilitating efficient repair of DSBs via c-NHEJ. (K) Schematic illustrating that ZBTB24 loss leads to a shift from c-NHEJ to a-NHEJ and impaired CSR at AID-induced DSBs in B cells.

Article Snippet: Briefly, IP samples were resolved on 8% denaturing PAGE gels along with purified human PARP1 (Aparptosis) as a positive control.

Techniques: Transfection, Expressing, Plasmid Preparation, Amplification, Western Blot, Clone Assay, Control, Flow Cytometry, Transduction, Infection