Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1

Abstract

53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.

Figure 1. 53BP1 effects on joining efficiency of proximal (1 kb) or distal (27 Mb) DSBs

(A) Schematic representation of IgH^I-1k allele before (top) and after (bottom) I-SceI induced recombination. I-SceI sites are indicated as blue circles, loxP sites as red triangles. Spacer sequence of 1.2 kb is indicated as yellow rectangle. (B) Bar graph shows I-SceI induced recombination frequency of IgH^I-1k/+AID^−/− B cells in the presence or absence of 53BP1. P value was calculated using a paired two tailed students t-test. See also panel S1F. (C) Left: Bar graph showing the frequency of I-SceI induced recombination products with more than 35 nts end processing for IgH^I-1k/+AID^−/− and IgH^I-1k/+AID^−/−53BP1^−/− B cells. Right: Dot plot showing resection in sequences from I-SceI infected IgH^I-1k/+AID^−/− and IgH^I-1k/+AID^−/−53BP1^−/− B cells, with each dot representing one cloned sequence. (D) Schematic representation of IgH^I-27M allele before (top) and after (bottom) I-SceI induced recombination. (E) As in (B) for IgH^I-27M/+AID^−/− and IgH^I-27M/+AID^−/−53BP1^−/− B cells. See also panel S1G. (F) As in (C) for IgH^I-27M/+AID^−/− and IgH^I-27M/+AID^−/−53BP1^−/− B cells. (G) Schematic representation of the Myc^I and IgH^I alleles. (H) As in (B and E) for IgH^I/+Myc^I/+AID^−/− B cells in the presence and absence of 53BP1. See also panel S1H. In all figures: Horizontal lines in dot plots indicate the means; Error bars indicate standard deviations; p values were calculated using a two-tailed students t-test, unless otherwise indicated; All graphs represent data from at least three independent experiments, unless specified. See also Figure S1.

Figure 2. The BRCT domains are dispensable for CSR and DNA end protection

(A) Schematic representation of wild type (WT) 53BP1 protein (top) and 53BP1 lacking the BRCT domains (bottom). (B) Western blot showing 53BP1 expression levels in WT and 53BP1^ΔBRCT B cells. (C) Left: Representative flow cytometry plots measuring CSR after stimulation of WT, 53BP1^ΔBRCT and 53BP1^−/− B cells. Numbers indicate the percentage of IgG1 switched cells. CFSE dye tracks cell division. Right: Summary dot plot indicating CSR as a percentage of WT value within the same experiment. Each dot represents an independent experiment. (D) Left: Representative ethidium bromide stained argarose gels showing PCR products obtained after I-SceI induced recombination in IgH^I-96k/+, IgH^I-96k/+53BP1^ΔBRCT/− and IgH^I-96k53BP1^−/− B cells. Middle: Bar graph quantitating the frequency of I-SceI induced recombination products with more than 35 nts end processing. Error bars indicate standard deviation. Right: Dot plot showing resection with each dot representing one sequence. Two independent experiments. See also Figure S2.

Figure 3. 53BP1 tudor domains are required for CSR and for the protection of DNA ends

(A) Western blots of fractionated WT B cells +/− 10 Gy of IR. CYTO, cytoplasmic fraction; NS, nuclear soluble fraction; CHR, chromatin fraction. (B) Schematic representation of WT 53BP1 (top) and 53BP1 with tudor domain mutation D1518R (bottom). (C) 53BP1 and γ-H2AX IRIF in WT and 53BP1^DR MEFs after IR. (D) Western blots of unstimulated, fractionated WT and 53BP1^DR B cells. (E) Left: Representative flow cytometry plots measuring CSR to IgG1 after stimulation of WT, 53BP1^DR and 53BP1^−/− B cells. Right: Summary dot plot indicating CSR as a percentage of WT. Each dot represents an independent experiment. (F) As in Figure 1(C) for WT, 53BP1^DR and 53BP1^−/− B cells. Error bars indicate standard deviation. Two independent experiments. See also Figure S3.

Figure 4. 53BP1 chromatin association in the absence of H2AX is not sufficient to prevent end resection

(A) Western blots of fractionated AID^−/− and AID^−/−H2AX^−/− B cells. (B) Left: Bar graph showing the frequency of I-SceI induced recombination products with more than 35 nts end processing for IgH^I-96k/+AID^−/−, IgH^I-96k/+AID^−/−53BP1^−/− and IgH^I-96k/+AID^−/−H2AX^−/− B cells. Right: Dot plot showing resection in sequences from I-SceI infected IgH^I-96k/+AID^−/−, IgH^I-96k/+AID^−/−53BP1^−/− and IgH^I-96k/+AID^−/−H2AX^−/− B cells. Error bars indicate standard error of the mean. Two independent experiments. (C) Histogram with number of radial structures in metaphases from PARP inhibitor treated Brca1^lox/loxCD19^cre/+ (= Brca1^mutant) B cells either proficient or deficient for H2AX. Error bars indicate standard error of the mean. Two independent experiments.

Figure 5. The oligomerization domain of 53BP1 in chromatin association and CSR

(A) Schematic representation of WT 53BP1 (top) and 53BP1 lacking amino acids 1210–1447 (bottom). (B) As in Figure 2(C) for WT, 53BP1^{Δ1210–1447} and 53BP1^−/− B cells. (C) Diagram of 53BP1 retroviral constructs with the indicated mutations and deletions. (D) As in Figure 2(C) after infection of 53BP1^−/− B cells with empty retrovirus, or retrovirus expressing 53BP1^1–1710, or the oligomerization mutant 53BP1^{Δ1231–1270}, or the other mutants listed in panel (C). (E) Western blots of fractionated 53BP1^−/− B cells stimulated and infected with 53BP1^1–1710 or 53BP1^{Δ1231–1270}. (F) Western blots of fractionated 53BP1^−/− B cells stimulated and infected with 53BP1^1–1710 or 53BP1^1052–1710. (G) As in Figure 2(C) after infection of 53BP1^−/− B cells with empty retrovirus, or retroviruses expressing 53BP1^1–1710 or the N-terminal deleted mutant 53BP1^1052–1710. See also Figure S4.

Figure 6. N-terminal phosphorylation of 53BP1 in DNA damage and CSR

(A) Diagram of 53BP1 retroviral constructs with the indicated mutations. (B) As in Figure 2(C) after infection of 53BP1^−/− B cells with empty retrovirus, or retroviruses expressing 53BP1^1–1710, or the N-terminal mutant 53BP1^28A, or the other mutants listed in panel (A). (C) Western blots of fractionated 53BP1^−/− B cells stimulated and infected with 53BP1^1–1710 or 53BP1^28A. (D) 53BP1 IRIF in 53BP1^−/− MEFs reconstituted with 53BP1^1–1710 and 53BP1^28A after 10 Gy. See also Figure S5.

Figure 7. Domains of 53BP1 required for preventing DNA end resection

(A) Brca1^Δ11/Δ1153BP1^−/− B cells reconstituted with 53BP1 mutant retroviruses. Top: examples of normal metaphases (+empty) or metaphases containing radial chromosome structures (+53BP1^1–1710). Bottom: histogram quantitating the number of radial structures upon infection with the indicated retroviruses. Error bars indicate standard error of the mean. Two independent experiments. (B) Table summarizing which functional domains of 53BP1 are required (R) or dispensable (D) for IRIF, chromatin binding, CSR and protection of DNA ends from resection. See also Figure S6.