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. 2020 Jul 9;9:e56008. doi: 10.7554/eLife.56008

Figure 1. Establishment of a compound screening platform for monitoring the efficiency of DSB repair by HR and NHEJ.

(A) Diagram of the HR-NHEJ dual fluorescent reporter (Chen et al., 2019). SD, splice donor; SA, splice acceptor. In the reporter cassette for simultaneous analysis of HR and NHEJ at the same chromosomal site, it contains two parts separated by a CMV promoter. The part downstream of the promoter contains two GFP exons separated by the engineered Pem1 intron with splice donor and acceptor, an adenoviral exon (AD2) with splice donor and acceptor, two I-SceI recognition sites in an inverted orientation, and an ATG-less tdTomato gene. The second part upstream of the promoter contains the Pem1 intron and a full-length tdTomato gene with a splice donor before start codon ATG. The homology region between the two parts of the reporter is approximately 3.9 kb including the rat Pem1 intron (marked in yellow line) and the tdTomato gene. (B) Schematic diagram of the doxycycline-inducible I-SceI expression vector. rtTA, reverse tetracycline trans-activator protein; PTRE, promoter of tetracycline response element; PCAG, the CAG promoter, a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian systems. (C–D) Generation of the compound screening platform. The linearized doxycycline-inducible I-SceI expression vector was nucleofected into D4a cells followed by selection with 30 μg/mL hygromycin B. Then individual colonies were picked and one of the colonies, CLZ3, was utilized for further studies. I-SceI endonuclease expression could be efficiently induced upon doxycycline supplementation (C), further resulting in the generation of DSBs on the reporter cassette. Successful repair by HR or NHEJ results in functional tdTomato or GFP, turning cells red or green respectively, which could be observed via microscopy (D). (E) Flow cytometry analysis of tdTomato+ and GFP+ cells upon doxycycline addition (left panel). The representative FACS traces are shown (right panel). (F) Workflow of compound screening using the CLZ3 cell line. (G) 722 small molecules were screened using CLZ3 cells and farrerol (highlighted in blue) was identified as an HR enhancer.

Figure 1—source data 1. The list of small molecule compounds and their influences on DNA repair.
Figure 1—source data 2. Summary of NHEJ, HR and SSA efficiency.

Figure 1.

Figure 1—figure supplement 1. Farrerol stimulates the precise HR in human fibroblasts and mouse ESCs.

Figure 1—figure supplement 1.

(A) Farrerol significantly promotes HR efficiency in HCA2-H15c cells harboring the single HR reporter (left panel), while does not influence NHEJ efficiency in HCA2-I9a cells harboring the single NHEJ reporter (right panel). The HR and NHEJ reporters were generated as previously described (Mao et al., 2008a). SD, splice donor; SA, splice acceptor; the yellow line stands for the homologous arm. (B) Farrerol significantly enhances HR efficiency in mouse ESCs. HR reporter was linearized in vitro with I-SceI to induce DSBs. Mouse ESCs were pretreated with indicated doses of farrerol for 24 hr. Then 0.5 μg purified linearized HR reporter was transfected into 2 × 105 mouse ESCs along with 100 ng plasmid encoding pDsRed2-N1 for normalizing the transfection efficiency. Farrerol was supplemented into the culture medium of transfected cells until FACS analysis at 48 hr post transfection. The ratio of GFP positive cells versus DsRed positive cells was used as the measure of HR efficiency. Error bars represent the s.d. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s., not significant, t-test. All experiments were repeated at least three times.
Figure 1—figure supplement 2. Farrerol accelerates the recruitment of RAD51 rather than influences the expression of the indicated HR-related factors.

Figure 1—figure supplement 2.

(A) Farrerol does not influence the protein level of important HR factors, and 53BP1, a critical negative regulator of HR. (B) Analysis of cell cycle distribution in HCA2-hTERT cells treated with farrerol at indicated concentrations. (C–D) Farrerol does not affect the recruitment of RPA2 in HCA2-hTERT cells post 2 Gy X-Ray treatment. (E–F) Farrerol promotes RAD51 recruitment in HCA2-hTERT cells post 2 Gy X-Ray treatment. Error bars in (B) represent the s.d. Error bars in (D) and (F) represent the s.e.m. **p<0.01, ***p<0.001, ****p<0.0001, t-test.
Figure 1—figure supplement 3. Farrerol inhibits the SSA efficiency.

Figure 1—figure supplement 3.

(A) Schematic diagram of reporter cassette HRF measuring gene conversion, crossing over, and SSA. The cassette contains two copies of defective GFP-Pem1. The first copy has two inverted I-SceI recognition sites, and a 22-nt deletion on the first exon of GFP, and the second copy of GFP lacks the start codon. I-SceI induced DSBs can be repaired by gene conversion, crossing over or SSA, resulting in functional GFP. SD, splice donor; SA, splice acceptor; the yellow line stands for the homologous arm. (B) Schematic diagram of reporter cassette HRIF measuring gene conversion and crossing over. The positions of the two copies of defective GFP is different in HRF and HRIF. In HRF reporter, both copies are downstream CMV promoter while in HRIF reporter the CMV promoter is located between the two copies of GFP. In response to I-SceI digestion, only gene conversion or crossing over can reconstitute functional GFP. SD, splice donor; SA, splice acceptor; the yellow line stands for the homologous arm. (C) Effect of farrerol and RS-1 treatment on SSA efficiency. The two reporters were integrated into HCA2-hTERT cells, and a pool of colonies containing chromosomally integrated reporters were mixed for further analysis. HRF minus HRIF was employed as the measure of SSA efficiency. 5 μg I-SceI vector and 15 ng pDsRed2-N1 were transfected into reporter cells pre-treated with different small molecules. Small molecules were still added to the cells until FACS analysis three days after transfection. The ratio of GFP positive cells versus DsRed positive cells was used as the measure of repair efficiency. Error bars represent the s.d. *p<0.05, **p<0.01, ***p<0.001, n.s., not significant, t-test. All experiments were repeated at least three times.