Mice
All mice were housed at the China Agricultural University Laboratory Animals Resource Center, where they were subjected to a standard light–dark cycle of 12 h each, and maintained at a temperature of 20–22 °C. All animal experiments were conducted in Sen Wu’s laboratory and were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Approval Number: SKLAB-2012-11). We have complied with all relevant ethical regulations for animal use. The 5 weeks old female ICR mice were procured from Beijing Vital River Laboratory Animal Technology.
Cell culture
The OG cell lines were maintained on feeder cells using a basic serum/LIF medium. This medium comprised DMEM (Gibco, 10829018), 15% FBS (Gibco, 10099), 1% penicillin/streptomycin (Gibco, 15104122), 1% non-essential amino acids (Gibco, 11140050), 1% GlutaMAX (Gibco, 35050079), 106 units/L of mouse LIF (Millipore, ESG1106) and 100 mM β-mercaptoethanol (Gibco, 15104122). The feeder cells were treated with mitomycin C (Amresco, MJ594) to inhibit their growth and were cultured in DMEM (Gibco, 11960) supplemented with 10% FBS (Gibco, 10099), 1% penicillin/streptomycin (Gibco, 15104122), 1% non-essential amino acids (Gibco, 11140050) and 1% sodium pyruvate (Gibco, 15104122). Other small molecules mentioned in our article were procured from Selleck and added individually to the basal serum/LIF medium at varying concentrations. Subculturing of all cell lines was performed every 2–3 days at a ratio ranging from 1:6 to 1:10 using Tryple (Gibco, 12605028). To establish the MERVL-tdTomato reporter cell lines, the OG cell lines underwent transfection via electroporation and were subsequently selected using 1 µg/ml puromycin for 7 days. Clones were then isolated and confirmed through polymerase chain reaction (PCR) analysis. All cell lines were maintained in a humidified incubator at 37 °C with 5% CO2.
Genome-wide CRISPR KO screens
The CRISPR-Cas9 DNA library contained 130,209 sgRNAs about 20,611 genes constructed by our laboratory. To obtain the mutant cell library, we transfected 108 OG cells with the MERVL reporter by electroporation (2B Nucleofector System, Lonza). After 24 h transfection, the cells were selected with 350 ng/µl G418 (InvivoGen, ant-gn-5) for 7 days. We expanded these cells to 3 × 108 for primary screen. We collected 2C-positive cells using FACS and amplified them for consecutive screen. Each round of screening was repeated three times. Medium needed to be changed every day.
Genomic DNA were extracted from 108 cells of the mutant cell pool and 5–10 × 106 cells of 2C-postive from the consecutive screen. The integrated sgRNA sequences were amplified by PCR using Gotaq DNA Polymerase (Mei5bio, MF002) with the left primer (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNTGAAAGTATTTCGATTTCTTGG-3′) and the right primer (5′- CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTTGATAACGGACTAGCCTTATT-3′). Then PCR products were purified and sequenced by Illumina HiSeq TM4000. We used MAGeCK package to analyze our screening results and took Z-score into consideration. Ggplot2 was used to summarize these results.
Generation of CRISPR KO cells
To generate CRISPR KO cells, we designed two sgRNAs for each target gene and cloned them into the pSg6 Plasmid. Subsequently, we transfected 106 mESCs using the Lonza 2B Nucleofector System. Twenty-four hours after transfection, cells were subjected to selection with 350 ng/µl G418 for a duration of 7 days. Following selection, individual clones were isolated into a 48-well plate, and their targeted regions were verified. Detailed sequences of the sgRNAs can be found in Table S1.
Overexpression of MDM2 in mESCs
Total RNA was extracted to amplify the CDS of Mdm2 by PCR. Subsequently, the amplified fragment was cloned into the PB vector under the control of the EF1α promoter. A total of 4 µg of the vector was transfected into 106 mESCs using the Lonza 2B Nucleofector System. Following transfection, mESCs were cultured for 24 h and then subjected to selection with 350 ng/µl G418 for a period of 7 days. Clones were subsequently isolated and expanded in a 48-well plate to allow for the extraction of RNA and protein.
RNA extraction and qPCR
Total RNA was extracted from cells using the RaPure Total RNA Kits (Magene, R4011) following the manufacturer’s protocol. Subsequently, 1 µg of RNA was reverse transcribed into cDNA using the ABScript III RT Master Mix (Abclonal, RK20429). qPCR was conducted using the 2x RealStar Green Power Mix (Genestar, A311-10) on a Roche PCR machine. The relative quantification of each gene was achieved by normalizing to Gapdh. A complete list of primers utilized for qPCR can be found in Table S2.
RNA sequencing and bioinformatics analysis
Total RNA was purified using magnetic beads with Oligo (dT) to selectively isolate mRNA. RNA-seq libraries were subsequently constructed and assessed using a combination of TIANGEN Biotech and the Agilent 2100 BioAnalyzer. The sequencing process generated 150 bp paired-end reads using PE150 on an Illumina platform. To analyze the data, clean reads were mapped to the Mus musculus genome using HISAT2. Read counts were quantified using HTSeq-count (v0.6.0) and then normalized to obtain the Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) values. Differentially expressed genes (DEGs) were identified using edgeR with criteria including an absolute log2 (fold change) > 2 and a p value < 0.05. Subsequently, the datasets were further analyzed using the R programming language.
Flow cytometry
Cell sorting and analysis were carried out using the FACS CaliburTM flow cytometer (BD, San Jose, CA, USA). Data visualization was conducted using FlowJo software version 10. For our gating strategy, we employed wild-type (WT) mESCs with the MERVL reporter.
Co-immunoprecipitation
Collect 293T cells cultured in 10 cm culture dishes using 1 mL of IP lysis buffer (Beyotime, P0037), and transfer them to 1.5 mL centrifuge tubes. Let them lyse on ice for 30 min. After centrifugation at maximum speed (4 °C, 21,100 g, 10 min), collect the supernatant for subsequent Co-IP experiments. Supernatants were incubated with Pierce™ beads (Thermo Scientific™, 88802) for 6–8 h at 4 °C with rotation. Antibodies used include FLAG (Sigma, F1804, 1:200 mouse) and HA (Beyotime, AH158, 1:200 mouse), IgG (Beyotime, A7028, 1:200 mouse). The protein solution collected was denatured at 95 °C for 10 min using 10% SDS–PAGE for western blot analysis.
Western blotting
The cells were lysed using IP lysis buffer supplemented with 100× PMSF and incubated on ice for 30 min. The supernatant was obtained by centrifuging at 4 °C and 20,000 × g for 15 min. To determine protein concentration, we utilized the BCA protein assay kit (Beyotime, P0012) according to the manufacturer’s instructions. Equal amounts of protein were denatured using 10% SDS–PAGE. For western blotting, the following primary antibodies were used: MDM2 (Abcam, ab259265, 1:1000 rabbit), SUZ12 (Cell Signal Technology, 3737, 1:1000 rabbit), ACTIN (Beyotime, AA128, 1:1000 mouse), TUBULIN (Beyotime, AF2835, 1:1000 mouse), HA (Beyotime, AH158, 1:1000 mouse), FLAG (Sigma, F1804, 1:1000 mouse), H3 (Elabscience, E-AB-22003, 1:1000 mouse) and H3K27me3 (Sigma, 07-449, 1:10,000 rabbit).
Immunofluorescence
Embryos were subjected to fixation with 4% paraformaldehyde for a duration of 30 min and subsequent permeabilization using 0.5% Triton X-100 for 30 min at room temperature. Blocking of embryos was treated with 1% BSA in PBS supplemented with 0.1% Tween 20, lasting for 60 min at room temperature. Incubation with CDX2 primary antibodies (Biogenex, MU392A, 1:200 mouse) occurred overnight at 4 °C, followed by incubation with secondary antibodies (Invitrogen, A32723, 1:500) for 1 h at room temperature. Lastly, the nuclei of the embryos were stained with DAPI (Beyotime, P0131), and images were acquired using the fluorescence microscope.
Cell cycle analysis
Cells were fixed by incubating with 70% ethanol at −20 °C overnight. The next day, the fixed cells were centrifuged at 4 °C, 300 × g, and washed once with PBS. Afterward, RNase A treatment was performed at 37 °C for 30 min. Finally, the cells were stained with propidium iodide (PI) at 4 °C for 30 min. Cell cycle analysis was performed using a BD flow cytometer, and the data were analyzed with Modfit LT software to determine the distribution of cells across different phases of the cell cycle.
Nutlin-3 treatment
To inhibit the expression of Mdm2 in pre-ZGA embryos, zygotes were cultured in KSOM or HM medium supplemented with Nutlin-3 (Selleck, S1061) at a final concentration of 5 μM. DMSO was included as a negative control. The embryos were cultured at 37 °C and monitored daily until they reached the blastocyst stage.
CUT&Tag
CUT&Tag assay was conducted using the NovoNGS CUT&Tag 3.0 High-Sensitivity Kit (Novoprotein, N259). Approximately 1 × 105 mESCs were incubated with 10 μL of Binding ConA beads. Primary antibodies targeting FLAG (Sigma, F1804, 1:50 mouse) and H3K27me3 (Sigma, 07-449, 1:100, rabbit) were incubated overnight at 4 °C. Subsequently, secondary antibodies were added, and the mixture was incubated at room temperature for 1 h. Following this, the samples were treated with 1 μL of Transposome and incubated at room temperature for 1 h. DNA was then collected for subsequent PCR analysis. The libraries were amplified and subjected to sequencing using the Illumina NovaSeq PE150 platform following the manufacturer’s instructions.
ChIP-seq and CUT&Tag data analysis
The raw data underwent quality filtering using Trimmomatic to obtain clean data. These clean data were then aligned to the mm10 genome using Bowtie2. For the identification of peaks, we utilized MACS2. Heatmaps were generated using Deeptools, and ChIPseeker was employed to annotate the promoters.
Public datasets reanalyzed
We conducted a re-analysis of publicly available datasets. H3K27me3 ChIP-seq data for mESCs and SUZ12 KO mESCs were obtained from GSE103685. Additionally, H3K27me3 modification data for 2C embryos were acquired from GSE73952, and RNA-seq data for mouse and pig pre-implantation embryos were retrieved from GSE71434 and GSE163709.
Statistics and reproducibility
The statistical differences were analyzed by the Student’s t-test when two independent groups were compared. Data are displayed in a bar graph with error bars representing the mean ± SD and individual sample points shown. GraphPad Prism was used for the statistical analysis of data. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Three independent biological replicates were included and the figure legends specify the sample sizes.
Reporting summary
Detailed information on the research design can be found in the Nature Portfolio Reporting Summary associated with this article.
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- Source: https://www.nature.com/articles/s42003-024-06507-9