Spartan/C1orf124, a reader of PCNA ubiquitylation and a regulator of UV-induced DNA damage response

Abstract

PCNA is a key component of DNA replication and repair machineries. DNA damage-induced PCNA ubiquitylation serves as a molecular mark to orchestrate postreplication repair. Here, we have identified and characterized Spartan, a protein that specifically recognizes ubiquitylated PCNA and plays an important role in cellular resistance to UV radiation. In vitro, Spartan engages ubiquitylated PCNA via both a PIP box and a UBZ domain. In cells, Spartan is recruited to sites of UV damage in a manner dependent upon the PIP box, the UBZ domain, and PCNA ubiquitylation. Furthermore, Spartan colocalizes and interacts with Rad18, the E3 ubiquitin ligase that modifies PCNA. Surprisingly, while Spartan is recruited by ubiquitylated PCNA, knockdown of Spartan compromised chromatin association of Rad18, monoubiquitylation of PCNA, and localization of Pol η to UV damage. Thus, as a "reader" of ubiquitylated PCNA, Spartan promotes an unexpected feed-forward loop to enhance PCNA ubiquitylation and translesion DNA synthesis.

Figure 1. Spartan Is Important for the Cellular Response to UV-Induced DNA Damage

(A) A depiction of the conserved domains of Spartan. (B and C) Spartan knockdown cells are sensitive to UV, but not IR. U2OS cells transfected with control, Spartan, Rad18, or ATM siRNA were treated with the indicated doses of UV (B) or IR (C). Cell viability was determined as described in the Experimental Procedures section. ATM knockdown cells are hypersensitive to IR and serve as a positive control in (C). Error bars: SD, n ≥ 2. (D) Spartan colocalizes with PCNA at sites of UV-induced DNA damage. U2OS cells stably expressing SFB-Spartan were treated with 50J/m² UV or left untreated. Cells were subsequently extracted with Triton at 1 hr, fixed, and stained with the indicated antibodies. (E) Spartan does not localize to sites of DSBs. Cells stably expressing SFB-Spartan were treated with 2 Gy IR and analyzed with the indicated antibodies.

Figure 2. Spartan Contains a Functional UBZ Domain

(A) Sequence alignment of the UBZ domain of Spartan across different species. Green denotes important residues for ubiquitin binding; blue denotes residues mutated in the ΔUBZ mutant used in this study. Note that X. laevis uniquely contains two UBZ domains. (B) SFB-Spartan^WT and SFB-Spartan^ΔUBZ were analyzed by western blot. (C) Spartan is ubiquitylated in a UBZ-dependent manner. SFB-Spartan^WT was isolated under denaturing conditions and analyzed with the indicated antibodies. (D) Spartan’s UBZ domain binds ubiquitin chains directly. Purified GST, GST-Spartan-C^WT, GST-Spartan-C^ΔPIP, and GST-Spartan-C^ΔUBZ were incubated with purified K63- or K48-linked ubiquitin chains and subsequently retrieved with glutathione beads. The ubiquitin chains in input and pull-downs were analyzed using K63- or K48-Ub-specific antibodies.

Figure 3. Spartan Binds Ubiquitylated PCNA via Its PIP Box and UBZ

(A) Sequence alignment of the PIP box of Spartan across different species. *: putative PIP boxes in locations different from the PIP box of human Spartan. Red denotes important residues for PCNA binding; blue denotes residues mutated in the ΔPIP mutant used in this study. (B) Spartan’s PIP box and UBZ domain are important for efficient PCNA binding in vitro. 293T cells were treated with 50J/m² UV and lysed after 5 hr. GST, GST-Spartan-C^WT, GST-Spartan-C^ΔPIP, GST-Spartan-C^UBZ, and GST-Spartan-C^ΔPIP,UBZ were incubated with cell extracts and subsequently retrieved with glutathione beads. The PCNA in input and pull-downs was analyzed by western blot. (C) Quantification of the relative abundance of unmodified and ubiquitylated PCNA in Figure 3B. (D) Spartan’s PIP box is important for PCNA binding in cells. 293T cells were transiently transfected with empty vector, SFB-Spartan, or SFB-Spartan^ΔPIP. Cell extracts were subjected to streptavidin-coated Dynabead pull-down, followed by western blot.

Figure 4. The PIP Box and UBZ Domain of Spartan Are Important for Its Function In Vivo

(A) Colocalization of Spartan with PCNA requires both its PIP box and UBZ domain. U2OS cells stably expressing SFB-Spartan^WT, Spartan^ΔPIP, or Spartan^ΔUBZ were irradiated with UV through 5-micron filters and subsequently analyzed by immunofluorescence after 1 hr as in Figure 1D. (B) Effects of Rad18 and Usp1 knockdown on Spartan focus formation. Cells expressing SFB-Spartan^WT were transfected with control, Rad18, or Usp1 siRNA. Three days after transfection, cells were irradiated with UV and analyzed as in (A). Quantification of the percentage of PCNA-positive cells with Spartan foci is plotted. **: a p value of < 0.005; error bars: SD, >750 cells per condition. (C) Spartan’s PIP box and UBZ domain are important for normal UV resistance. Parental U2OS cells or derivatives stably expressing SFB-Spartan^WT, Spartan^ΔPIP, or Spartan^ΔUBZ were transfected with control, Spartan-1 (targeting the coding sequence; CDS), or Spartan-2 (targeting the 3′UTR) siRNA. Cell survival was analyzed after UV irradiation. Error bars: SD, n ≥ 2.

Figure 5. Spartan Regulates Localization of Pol η but Not XPA

(A) Spartan is necessary for the efficient recruitment of DNA polymerase η to sites of local UV-induced DNA damage. XP30RO cells stably expressing eGFP-Pol η were transfected with control, Spartan, or Rad18 siRNA, and irradiated with UV through 5-micron filters. After 5 hr, cells were extracted with Triton, fixed, and stained with antibodies against GFP and PCNA. (B) Quantification of the percentage of cells with PCNA-positive spots that are also positive for eGFP-Pol η. **:p < 0.05. Error bars: SD, n = 3 independent experiments each, in which >50 PCNA-positive spots were counted for each condition. (C) Spartan knockdown has no effect on the localization of XPA to sites of UV-induced DNA damage. U2OS cells were transfected with control or Spartan siRNA and UV-irradiated through filters. After 1 hr, cells were stained with antibodies for CPDs and XPA. The percentage of cells with CPD-positive spots that were also positive for XPA are plotted. Error bars: SD, n > 350 CPD spots counted for each condition.

Figure 6. Spartan Promotes UV-Induced PCNA Ubiquitylation

(A) Spartan is required for efficient UV-induced PCNA ubiquitylation. U2OS cells were transfected with control or three independent Spartan siRNAs. Three days after transfection, cells were treated with 50J/m² UV and analyzed after 5 hr. Levels of unmodified PCNA (PCNA) and monoubiquitylated PCNA (PCNA-Ub) were analyzed by western blot and quantified using ImageJ. (B) Defective UV-induced PCNA ubiquitylation in Spartan knockdown cells is observed over a range of UV doses. Cells stably expressing SFB-Spartan^WT were transfected with control or Spartan siRNA, irradiated with the indicated UV doses, and analyzed as in (A). (C) The defect of PCNA ubiquitylation in Spartan knockdown cells is suppressed by SFB-Spartan^WT. Cells stably expressing SFB-Spartan^WT were transfected with the indicated siRNAs and analyzed as in (A). (D) Spartan’s PIP box and UBZ domain are required for efficient PCNA ubiquitylation. Cells stably expressing SFB-Spartan^WT, Spartan^ΔPIP, or Spartan^ΔUBZ were transfected with Spartan-2 siRNA and were analyzed as in (A).

Figure 7. Spartan Is Functionally and Physically Linked to Rad18

(A) Spartan and Rad18 function in the same pathway to confer cellular UV resistance. U2OS cells were transfected with the indicated siRNAs, individually or in combination, and cell survival was measured after UV irradiation as described in the Experimental Procedures section. Error bars: SD, n ≥ 2. (B) Rad18 and Spartan colocalize at sites of UV damage. U2OS cells stably expressing SFB-Spartan^WT were nucleofected with GFP-Rad18 and irradiated with UV directly or through filters. After 1 hr, cells were extracted with Triton, fixed, and stained with GFP and FLAG antibodies. (C) Spartan and Rad18 physically interact with each other. 293T cells were transfected with plasmids expressing SFB-Spartan and GFP-Rad18, treated with UV or mock treated, and analyzed by IP after 1 hr. (D) Spartan is required for efficient chromatin association of Rad18. U2OS cells transfected with control or Spartan-2 siRNA were treated with UV or left untreated and subsequently subjected to chromatin fractionation after 5 hr. The indicated proteins in the chromatin and soluble fractions were analyzed by western blot. Orc2 serves as a loading control for the chromatin fractions.