Scientific Papers

ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells | Cell & Bioscience


Animals

The animals used in this project were maintained, cared, and used according to the animal protocol #NTU-105-EL-164 that was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University.

Mouse embryonic stem cells

The wildtype mESCs were derived from blastocyst embryos collected from fertilized superovulated female mice following our routine protocol [31].

To generate the pZscan4-GFP mESCs, the Zscan4 promoter sequence cloned from 2570 bp upstream of Zscan4c start codon [2] and a 720 bp eGFP coding sequence were cloned into the pSin vector (16578, Addgene) and the plasmid was transfected into HEK293T cell along with pSPAX2 (12260, Addgene) and pMD2.G (12259, Addgene) to produce lentivirus. Conditioned medium containing lentivirus was harvested and used to treat the wildtype mESCs, followed by FACS to enrich the pZscan4-GFP cells for subsequent culture.

To generate the Parp1 knockout mESC lines, we designed a guide RNA (gRNA, 5′-CTGGTACCATCCAACTTGCT-3′) targeting Exon 4 of the Parp1 gene. The gRNA was cloned to the Cas9 expressing plasmid (64221, Addgene) containing a mCherry reporter, following a reported CRISPR/Cas9 protocol [32]. We constructed a homologous recombination (HR) template containing a T2A-eGFP-stop codon sequence flanked by 1003 bp long homology arms on each side (Additional file 1: Fig. S3B). The Cas9 and HR template plasmids were transfected to mESCs by the lipofectamine stem transfection reagent (STEM00015, Thermo). 24 h after transfection, GFP and mCherry double positive mESCs were sorted out by FACS and single cell seeded in the 96-well plate to derive the KO clones. PCR (forward primer: GCCAGATGCGCCTGTCCA; reverse primer: TTCTTGATGGCCGGGAGCT) was performed to confirm the successful insertion.

The wildtype, pZscan4-GFP and the Parp1 KO mESCs were all cultured in Dulbecco’s modified Eagle’s medium (DMEM; 11965084, Thermo, Carlsbad, CA, USA) with 15% fetal bovine serum (FBS; TMR-016-B, Millipore, Darmstadt, Germany) supplemented with 1% Penicillin/Streptomycin Solution (P/S; 15140122, Thermo), 2 mM GlutaMax (35050061, Thermo), 0.1 mM nonessential amino acids (11140-050, Thermo), 0.1 mM 2-mercaptoethanol (ES-007-E, Millipore), 1 mM sodium pyruvate (11,360,070, Thermo) and 1000 units/mL Leukemia Inhibitory Factor (ESG1107, Millipore). Mitomycin C (2 μg/mL M4287, MilliporeSigma, Burlington, MA, USA) treated E13.5 mouse embryonic fibroblast (MEF) cells were used as the feeder cells for mESC culture.

HEK293T and BNL CL.2 cells

Human HEK293T (CRL-3216, ATCC, Manassas, VA, USA) and mouse BNL CL.2 (TIB-73, ATCC) cells were cultured in DMEM (11965084, Thermo) with 10% FBS (TMR-016-B, Millipore) supplemented with 1% P/S (15140122, Thermo). Plasmid transfection to these cells was performed by JetPrime (101000046, Polyplus, Illkirch-Graffenstaden, Bas-Rhin, France) following the manufacture’s instruction.

3-Aminobenzamide

3-Aminobenzamide (3-AB, A0788, MilliporeSigma) was dissolved in dimethyl sulfoxide (DMSO, D2650, MilliporeSigma) to the final concentration of 10 M as the stock solution. The stock solution was added to the culture medium at 2000 dilution to reach a working concentration of 5 mM 3-AB.

Immunofluorescence (IF) staining

Cells on cover slides were fixed with 10% formaldehyde (MA-H121-08, Crespellano, Italy). 2% bovine serum albumin (BSA, A9647, MilliporeSigma) and 0.25% Triton-X-100 (X100, MilliporeSigma) in phosphate-buffered saline (PBS, IB3012, Omics Bio, Taipei, Taiwan) was used for permeabilizing cells before they were incubated with the primary antibodies overnight at 4 ℃ followed by secondary antibodies and DAPI for 2 h at room temperature. The antibodies used were ZSCAN4 (ab4340, Millipore), FLAG (F7425, MilliporeSigma), FLAG (66008-4-Ig, Proteintech, Rosemont, IL, USA), γH2AX (ab2893, Abcam, Cambridge, UK), HA (sc-7392, Santa Cruz, Dalla, TX, USA), Alexa anti-mouse 488 (A11001, Thermo), Alexa anti-rabbit 488 (A11034, Thermo), Alexa anti-mouse 594 (A11032, Thermo), and Alexa anti-rabbit 647 (A27040, Thermo). The images were captured by the laser-scanning confocal microscope (TCS SP5 II confocal microscope, Leica, Wetzlar, Germany).

γH2AX foci counting

To count the number of γH2AX foci, images obtained from confocal microscopy were analyzed by the ImageJ software [33] (exampled in Additional file 1: Fig. S5). The counted number of cells in each experiment (range from 129 to 846) were listed in Additional file 1: Table S2.

Immunoprecipitation

Immunoprecipitation (IP) was performed by using the Dynabeads protein G IP kit (10007D, Thermo), following the manufacturer’s instruction. Briefly, HEK293T cells transfected with epitope-tagged expression plasmid(s) were lysed in the RIPA buffer (92590, Millipore) for 10 min at 4 ℃ and centrifuged at 16,000×g for supernatant collection. The Dynabeads were incubated with 4 μg HA antibody (sc-7392, Santa Cruz) or 2 μg FLAG antibody (F7425, MilliporeSigma) at room temperature for 10 min. Next, the Dynabeads were incubated with 500 µg cell lysate at 4 ℃ for 2 h. After washing, the IP samples were collected and used for Western blot (see next session) to complete the Co-IP assay. Appropriate host of IgG served as control which include mouse IgG (550878, BD, Franklin Lakes, NJ, USA) and rabbit IgG (550875, BD).

Western blot

For western with IP samples (see previous session), we included the input control which consists of 1% cell lysate. For regular western, 30 µg protein lysate from each sample was used.

Samples were run in electrophoresis using 10% acrylamide gels and then transfer to 0.22 µm PVDF membrane (GE10600021, Millipore). 5% skim milk in TBST (0.1% Tween 20 in TBS) was used to block the membrane for 30 min at room temperature. The membrane was immunoblotted with the primary antibody overnight at 4 ℃ then incubated with HRP-conjugated secondary anti-mouse antibody (31430, Thermo) or HRP-conjugated secondary anti-rabbit antibody (31460, Thermo) for 2 h at room temperature. The signal was detected by T-Pro LumiFast Plus Chemiluminescent Substrate Kit (JT96-K002, T-pro, New Taipei, Taiwan) and captured by GeneGnome XRQ Chemiluminescence with CCD (SynGene, Cambridge, UK). The primary antibodies included FLAG antibody (F7425, MilliporeSigma), HA (sc-7392, Santa Cruz), γH2AX (ab2893, Abcam), ZSCAN4 (ab4340, Millipore), and PARP1 (9542, Cell Signaling, Danvers, MA, USA).

Vector construction

Plasmids were constructed by the Gibson Assembly Master Mix (E2611L, New England BioLabs, Ipswich, MA, USA) using the pSin vector (16578, addgene, Watertown, MA, USA).

Statistical analysis

All quantitative data were represented as mean ± standard error of the mean (SEM), with at least 3 biological independent replicates. The statistical comparison between two groups was conducted by unpaired two-tailed student’s t-test (Numbers, Apple, Cupertino, CA, USA).



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