Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand breaks

Abstract

Cellular responses to DNA damage involve the relocalization of checkpoint proteins to DNA double-strand breaks (DSBs). The fission yeast checkpoint mediator protein Crb2, a homolog of mammalian 53BP1, forms ionizing radiation-induced nuclear foci (IRIF). The IRIF formation by Crb2 requires histone H2A C-terminal phosphorylation and H4-K20 methylation. However, the relevance of Crb2 relocalization is uncertain, because neither histone modification is required for a checkpoint response. Here we show that these histone modifications cooperate in the same Crb2 recruitment pathway, which also requires the Tudor and BRCT motifs in Crb2. In the absence of these histone modifications, an alternative recruitment pathway is sufficient for checkpoint activation and accumulation of Crb2 at a persistent DSB generated by HO endonuclease. This parallel pathway requires a cyclin-dependent kinase phosphorylation site in Crb2 that mediates an association with another BRCT protein Cut5 (the TopBP1 homolog), which also accumulates at HO-induced DSBs. We propose that such dual recruitment mechanisms may be a common feature of DNA damage checkpoint mediators.

Figure 1.

Histone H4-K20 but not H3-K79 is required for efficient Crb2 IRIF formation. (A) H4-K20R mutation dramatically reduced the number of IR-induced Crb2 foci, whereas mutations at three other conserved lysines in histone H3 and H4 had no effect. Cells with YFP-tagged Crb2 were treated with a 36-Gy γ irradiation and examined by fluorescence microscopy before and at different time points after irradiation. On average, cells receive about four DSBs at this dose (Du et al. 2003). Strains used were LLD3614, LLD3615, LLD3616, and TMN3617. Bar, 5 μm. (B) Quantitation of the microscopy results of the same experiment as in A. About 300 nuclei were scored for each time point.

Figure 2.

Eliminating both H4-K20 methylation and H2A phosphorylation causes weaker defects in DNA damage responses than crb2Δ Data shown are representative of multiple experiments. (A,B) H4-K20R mutation (h4.2K20R) and H2A C-terminal phosphorylation site mutations (hta-AQ), either alone or together, resulted in weaker sensitivity to UV, HU, and IR than crb2Δ Strains were constructed in a genetic background in which two of the three pairs of H3/H4 genes were deleted. Strains used were LLD3618, LLD3619, LLD3620, LLD3621, and LLD3622. (A) Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. Photos were taken 3 d later for untreated and UV-treated plates. The HU plate was photographed 5 d later. (B) Survival curves of cells treated with different doses of γ irradiation in liquid YES medium. Cells were plated on YES plates after treatment and incubated at 30°C. The numbers of colonies were counted after 3 d. (C,D) Deleting the H4-K20 methyltransferase gene (set9Δ) and mutating H2A C-terminal phosphorylation sites (hta-AQ), either alone or together, resulted in weaker sensitivity to UV, HU, camptothecin (CPT), and IR than crb2Δ Strains were constructed in a genetic background with full dosage of H3 and H4 genes. Strains used were LLD3431, LLD3623, TMN3291, LLD3624, LLD3259, LLD3625, LLD3626, and LLD3627. (C) Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. Plates were photographed 2 d later, except for the 5-mM HU plate, which was photographed 5 d later. (D) Survival curves of cells treated with different doses of IR or UV. The numbers of IR-surviving colonies were counted after incubation on YES plates at 30°C for 2 d. The numbers of UV-surviving colonies were counted after incubation on YES plates at 30°C for 3 d. (E) set9Δ and hta-AQ, either alone or together, resulted in a weaker checkpoint defect than crb2Δ Strains harboring the cdc25-22 mutation were synchronized at the G2 phase of the cell cycle by shifting to 35.5°C for 2.5 h before IR treatment. After IR treatment, cells were released back to 25°C medium. The progression through mitosis was monitored by DAPI and Calcofluor staining. The crb2Δ strain was not used in the 540 Gy experiment. Strains used were LLD3628, LLD3629, LLD3630, LLD3631, and LLD3632.

Figure 3.

A conserved Tudor domain in Crb2 is necessary for efficient Crb2 IRIF formation and a Tudor domain mutation affects the same pathway as set9Δ with regard to sensitivities to genotoxic agents. (A) Two conserved regions of Crb2—a Tudor domain in the middle of the protein and a C-terminal BRCT tandem repeat—are each necessary for efficient IRIF formation. See Supplementary Figure 2 for images. Truncated Crb2 fragments were fused at their N terminus with YFP and expressed from the crb2⁺ promoter in a crb2Δ strain. (B) Mutating a conserved phenylalanine residue in the Tudor domain largely abolished Crb2 IRIF formation. Cells were treated with a 36-Gy γ irradiation and observed by fluorescence microscopy immediately after treatment. Strains used were LLD3642 and LLD3643. Bar, 5 μm. (C) The crb2-F400A mutation caused milder sensitivity to genotoxic agents than crb2Δ, exhibited no additive phenotype when combined with set9Δ, and enhanced the genotoxic sensitivity of the crb2-T215A mutant in a fashion similar to set9Δ Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. IR treatment was applied to liquid cultures and cells were then spotted on plates. Plates were photographed 2 d later, except for the 5-mM HU plate, which was photographed 3 d later. Strains used were LLD3431, LLD3423, LLD3644, LLD3645, LLD3646, LLD3647, LLD3648, and LLD3649.

Figure 4.

H4-K20 methylation, H2A C-terminal phosphorylation, and the conserved motifs in Crb2 are not required for recruiting Crb2 to an HO-induced DSB. (A) Expression of HO endonuclease in a strain harboring a single HO cleavage site generated DSBs in the majority of the cells. Twofold serial dilutions of genomic DNA were used as template for PCR with a pair of primers annealing to regions flanking the HO cleavage site. PCR amplifying from the unrelated act1 locus was used as control. (B,C) ChIP analysis demonstrated that TAP-tagged Crb2 is recruited to chromatin regions adjacent to the HO-induced DSB in both wild-type and the histone modification mutants. (B) Locations of the PCR primer pairs used for amplifying DNA regions adjacent to the HO cleavage site. (C) DNA from the input (∼0.003% of total) and the IgG bead-bound fraction (∼13% of total) was analyzed by multiplex PCR. Strains expressing YFP-Crb2 served as control for nonspecific binding of DNA to the IgG beads. Strains used were LLD3652, LLD3650, LLD3716, LLD3717, and LLD3718. (D) Eliminating either or both H4-K20 methylation and H2A C-terminal phosphorylation did not abolish HO-induced YFP-Crb2 nuclear focus formation. CFP-tagged Rad22 expressed in the same cells was used as an independent marker for HO-induced DSBs. Merges were overlays of fluorescence images (YFP in red, CFP in green) and bright-field image of the same field. Strains used were LLD3650, LLD3651, LLD3652, and LLD3653. Bar, 5 μm. (E,F) HO-induced Crb2 nuclear focus formation can occur independent of the Tudor domain and the BRCT tandem repeat. (E) F400 residue in the Crb2 Tudor domain is dispensable for the HO-induced focus formation by full-length Crb2 protein, but is required for the HO-induced focus formation by Crb2(355–778). CFP-tagged Rad22 expressed in the same cells was used as an independent marker for HO-induced DSBs. Strains used were LLD3654, LLD3655, and LLD3656. (F) An N-terminal fragment of Crb2 missing both the Tudor domain and the BRCT tandem repeat, Crb2(1–358), when fused with a leucine zipper motif, could form HO-induced foci in the absence of H4-K20 methylation and H2A C-terminal phosphorylation. The strain used was LLD3658.

Figure 5.

Histone modification-independent relocalization of Crb2 to DSBs requires the Crb2-T215 residue. (A) HO-induced focus formation by YFP-Crb2T215A was abolished by set9Δ or hta-AQ mutations. Strains used were LLD3659, LLD3660, and LLD3661. Arrowheads indicate Rad22 nuclear foci in cells undergoing nuclear division. Bar, 5 μm. (B) T215 is required for HO-induced focus formation by Crb2F400A. The strain used was LLD3662. (C) T215 is required for HO-induced focus formation by Crb2(1–358)–LZ. The strain used was LLD3663. (D) T215A mutation rendered Crb2(1–358)–LZ defective in conferring resistance to DNA damage, and this defect could be rescued by fusing the Crb2 fragment to Rad22. Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. IR treatment was applied to liquid cultures and cells were then spotted on plates. Plates were photographed 2 d later, except for the 5-mM HU plate, which was photographed 3 d later. Strains used were LLD3431, LLD3423, LLD3666, LLD3667, LLD3668, LLD3669, LLD3670, and LLD3671.

Figure 6.

Cut5 is involved in the histone modification-independent relocalization of Crb2 to DSBs. (A,B) The Crb2-T215 residue is important for an interaction between Crb2 and Cut5. (A) Pairwise yeast two-hybrid assays using the HIS3 reporter were performed between a Cut5 N-terminal fragment (amino acids 1–190) as bait and different Crb2 fragments as prey. Fivefold serial dilutions of cells were spotted on the indicated plates. Plates supplemented with 2.5 mM 3-aminotriazole (3-AT) allow growth only when bait and prey interact. (B) Pairwise yeast two-hybrid assays using the lacZ reporter were performed between a Cut5 N-terminal fragment (amino acids 1–190) as bait and different Crb2 fragments as prey. The values represent the means of measurements on three independent transformants. Error bars represent the standard errors of the means. (C,D) Cut5 colocalizes with Crb2 at an HO-induced DSB. (C) Both Crb2 and Cut5 formed HO-induced nuclear foci and their foci overlapped with each other. (D) Immediately after a 36-Gy IR treatment, Crb2 nuclear foci were readily detectable, whereas Cut5 foci occurred at much lower frequency. Only a single Z-section was captured for the fluorescence images in these panels. Bar, 5 μm. The strain used was LLD3672. (E–I) The cut5-T401 mutation largely abolished Set9-independent HO-induced Crb2 focus formation. (E) HO-induced Crb2 foci in wild-type cells. (F) HO-induced Crb2 foci in set9Δ cells. (G) HO-induced Crb2 foci in cut5-T401 cells. (H) The lack of HO-induced Crb2 foci in set9Δ cut5-T401 double-mutant cells. However, these cells did elongate upon HO-induction. (I) Quantitation of the microscopy results of the same experiment as shown in E–H. About 200 Rad22 foci were counted for each strain. Strains used were LLD3650, LLD3651, LLD3673, and LLD3674. (J) The cut5-T401 mutation displayed a synthetic genetic interaction with set9Δ Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. IR treatment was applied directly to the plate. Plates were photographed 2 d later. Strains used were LLD3431, LLD3423, LLD3645, LLD3644, LLD3675, LLD3648, LLD3676, and LLD3677.