Elevated microRNA-187 causes cardiac endothelial dysplasia to promote congenital heart disease through inhibition of NIPBL

Sex as a biological variable. Our study examined male and female animals, and similar findings are reported for both sexes.

Human tissue samples. Our subjects were fetuses miscarried at about 20 weeks with nonsyndromic TOF (i.e., no 22q11.2 deletion; n = 5), and sex- and age-matched miscarried fetuses without TOF (n = 5) were used as controls. The diagnosis was obtained by echocardiography and confirmed during an anomaly scan. Written informed consent was obtained from a parent or legal guardian after they reviewed the consent document and had their questions answered. The right ventricle, brain, liver, lung, and kidney tissues were surgically excised from miscarried fetuses with TOF or control. All experiments in this study were conducted with approval from the Medical Ethics Committee at the Obstetrics and Gynecology Hospital of Fudan University.

Endothelial cell MACS separation. Single-cell suspensions of human and mouse hearts were prepared through tissue mincing and enzymatic digestion using an isolation enzyme kit (Thermo Fisher Scientific, 88281). Human and mouse hearts were collected and minced into approximately 1 mm blocks. Minced hearts were digested in 200 μL Isolation Enzyme 1 and Isolation Enzyme 2 (Thermo Fisher Scientific, 88281) in HBSS (Thermo Fisher Scientific, 88281) at 37°C for 30 minutes with tissue suspension triturated every 10 minutes. Five hundred microliters cold buffer consisting of 0.5% FBS (Corning, CGR-35-081-CV) and 2 mM EDTA (Invitrogen, AM9260G) in PBS (Gibco, 10010049) was added to stop digestion, and the resulting cell suspensions were filtered through a 40 μm cell strainer (Falcon, 352340) before centrifugation at 300g for 5 minutes at 4°C. Cell numbers were determined, and cell pellets were resuspended in 80 μL of buffer per 107 total cells. Twenty microliters of CD144 (VE-Cadherin) MicroBeads, human (Miltenyi Biotec, 130-097-857), or CD31 MicroBeads, mouse (Miltenyi Biotec, 130-097-418), were added for human hearts or mouse hearts, respectively. Mix well and incubate for 15 minutes at 4°C. Cells were washed by addition of 1 mL buffer and centrifuged at 300g for 5 minutes at 4°C. For detection of positive cell rate, the cell pellet was resuspended in 500 μL buffer, and human CD31-APC (eBioscience, 17-0319-42) or mouse CD31-APC (eBioscience, 17-0311-82) staining antibody was added before incubation for 15 minutes in the dark at 4°C. LS Columns were placed in a MACS separator (Miltenyi Biotec, 130-042-303) and rinsed 3 times with 1 mL of buffer. The cell suspension was added to the column and washed 3 times with 1 mL of buffer before the magnetically labeled cells were flushed out by firm pushing of the plunger into the column.

Cardiomyocyte separation. Collagenase II was used to dissociate mouse cardiac tissue at 37°C for 1 hour, followed by filtration through a 100 μm mesh to collect single-cell suspension. The suspension was treated with 1 mL of red blood cell lysis buffer at room temperature for 1 hour, centrifuged at 300g for 5 minutes to remove the supernatant, and subsequently incubated with 647 Mouse Anti–Cardiac Troponin T (BD Pharmingen, 565744) in PBS at 4°C for 30 minutes in the dark. Cardiac Troponin T–labeled cardiomyocytes were collected using the BD FACSAria cell sorter (BD Biosciences) and used for subsequent experiments.

Real-time RT-qPCR and RNA-Seq. Cells or tissue samples were extracted using TRIzol and isolated with a miRNeasy Mini Kit (QIAGEN, 217004) following the manufacturer’s recommendations. For miRNA detection, 2 μg of total RNA was used to synthesize cDNA using a miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, QP014). RT-qPCRs were next performed in 96-well plates using a miRNA RT-qPCR Detection Kit (GeneCopoeia, QP016). For mRNA detection, 500 ng of total RNA was used to synthesize cDNA using a HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R312). The mRNA levels were determined by RT-qPCR using HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, R333). All RT-qPCRs were performed using the Applied Biosystems QuantStudio 1 Real-Time PCR System in a volume of 20 μL. Data were quantified using the comparative Ct method, with U6 or GAPDH as a reference gene. Relative gene expression levels were calculated using the 2−ΔΔCt method. A list of the qPCR primers used in this study can be found in Supplemental Table 4.

Human endothelial cells (EA.hy926), hESC-ECs, and cardiac endothelial cells from P0 neonatal KI/KI and WT control mice were collected for RNA-Seq assay performed by BGI Genomics and APExBIO, respectively.

ATAC-seq and ATAC-qPCR. To prepare the sample for ATAC-qPCR, 50,000 viable cells were pelleted at 500 relative centrifugal force (RCF) at 4°C for 5 minutes, and the supernatant was aspirated. Next, 50 μL of cold ATAC–Resuspension Buffer (RSB) containing 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin was added to the cell pellet and pipetted up and down 3 times. The mixture was then incubated on ice for 3 minutes, and the lysis was washed out with 1 mL of cold ATAC-RSB containing 0.1% Tween-20 but no NP-40 or digitonin. The nuclei were pelleted at 500 RCF for 10 minutes at 4°C, and the supernatant was aspirated. The cell pellet was then resuspended in 50 μL of transposition mixture (25 μL 2× TD buffer (Vazyme, TD711-01), 2.5 μL transposase [100 nM final], 16.5 μL PBS, 0.5 μL 1% digitonin, 0.5 μL 10% Tween-20, 5 μL H2O) by pipetting up and down 6 times. The reaction was incubated at 37°C for 30 minutes. The DNA was subsequently purified with VAHTS DNA Clean Beads (Vazyme, N411-01) and amplified with barcode primers using the TruePrep DNA Library Prep Kit (Vazyme, TD501-01). Subsequent sequencing and data analysis were outsourced to APExBIO in Shanghai, China. ATAC-qPCR was performed using the same library construction method as in ATAC-seq. The ATAC libraries were subsequently adapted for RT-qPCR using specific primers designed based on previous articles (49).

CUT&Tag. The CUT&Tag assay used the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903). In brief, 105 endothelial cells were collected and washed with 500 μL of wash buffer. The cells were then bound to ConA beads for 10 minutes at 25°C. Subsequently, the cells were incubated with 1 μg of NIPBL (Bethyl Laboratories, A301-779A-T) or H3K27Ac (Abcam, ab177178) antibody at 4°C overnight. The next day, anti-rabbit IgG was added and incubated for 1 hour at 25°C. After 3 washes with DIG wash buffer (Vazyme, HD-102), the cells were incubated with 0.04 μM pA/G-transposon (pA/G-Tnp) for 1 hour at 25°C. After 3 washes with DIG 300 buffer, the cells were resuspended in tagmentation buffer and incubated at 37°C for 1 hour. Tagmentation was stopped by addition of proteinase K, buffer LB (Vazyme, HD-102), and DNA extract beads. The cells were then incubated at 55°C for 10 minutes, and the unbound liquid was removed after plating of the cells on a magnet. The beads were gently rinsed twice with 80% ethanol, and the DNA was eluted with double-distilled water. Libraries were constructed using the TruePrep Index Kit V2 for Illumina (Vazyme, TD202). Subsequent sequencing and data analysis were outsourced to GENEWIZ Biotechnology Co. Ltd.

Cell culture. Human embryonic stem cell line H9 (WA09, obtained from WiCell Research Institute) was cultured on Matrigel Matrix–precoated (1:200; Corning, 354277) 6-well plates at 10 μg/cm2 growth area in mTeSR1 Plus medium (StemCell Technologies, 100-0276) in a humidified incubator at 37°C with 5% CO2. The cells were seeded at a density of 5 × 105 cells per well, and the medium was replaced every 2 days.

EA.hy926 and HEK293T cells were obtained from the ATCC and cultured in DMEM (Gibco, 11995073) with 10% FBS (Corning, 35076111) and 1‰ Plasmocin (InvivoGen, ant-mpt) at 37°C and 5% CO2.

hESC-ECs were cultured in gelatin-coated (Sigma-Aldrich, G2500-100G) 6-well plates in EGM-2 Endothelial Cell Growth Medium-2 Bullet Kit (Lonza, CC-3162).

Differentiation of hESC-derived endothelial cells. The protocol to generate endothelial cells from hESCs was modified from the previously reported method (30). Briefly, hESCs were seeded on Matrigel-coated plates in mTeSR1 Plus medium to 30% confluence. At 30% confluence, the hESCs were pushed toward the mesodermal lineage by treatment with 6 μM CHIR-99021 (Selleck, S1263) in Essential 6 (E6; Gibco, A1516401) medium for 1 day, followed by a non-treatment in E6 medium for 1 day. At day 2 of differentiation, the cells were subjected to a differentiation medium composed of E6 medium supplemented with 300 ng/mL Recombinant Human VEGF (R&D Systems, 293-VE-010/CF), 200 ng/mL Recombinant Human FGF-2 (PeproTech 100-18B), 1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro; Sigma-Aldrich, 858463-25MG), and 50 μM melatonin (Sigma-Aldrich, M5250-1G) for 48 hours. From day 4 to day 12, the culture medium was changed every 48 hours into E6 medium supplemented with 10 ng/mL VEGF, 10 ng/mL bFGF, and 10 μM hydrocortisone (Selleck, S1696).

Lentivirus infection. To produce hESC-ECs expressing pri-miR-187 and/or NIPBL, human embryonic kidney (HEK) 293T cells were grown to 80% confluence on 100 mm plates. Cotransfection of 12 μg of pLJM1-pri-miR-187 and/or PCDH-NIPBL with the packaging plasmids (7.8 μg of pMDL, 6 μg of pREV, and 4.2 μg of pVSVG from Addgene) was carried out using Lipofectamine 2000 (Invitrogen, 11668019). After 48 hours, the viral supernatant was collected, concentrated using PEG 8000, and stored at –80°C. hESCs were infected with the pri-miR-187– and/or NIPBL-expressing lentivirus and then selected with puromycin (0.2 μg/mL; InvivoGen, ant-pr-1) for 2 weeks. RT-qPCR was performed to confirm the expression of miR-187 and NIPBL. The resulting hESC-ECs were pri-miR-187– and/or NIPBL-expressing hESC-ECs.

Immunofluorescence staining. Immunofluorescence staining was performed on hESC-ECs plated on Matrigel-coated glass-bottom dishes (NEST, Wuxi, China; 801001) or heart/HFO sections using a procedure previously described below in Formation and culture of HFOs. The cells or heart sections were fixed and permeabilized with 0.5% Triton X-100 (Sangon, A110694-0100) in PBS for 5 minutes and then blocked with 5% BSA (Sangon, A600332-0100) in PBS for 1 hour at room temperature. The samples were incubated with primary antibodies diluted in 3% BSA blocking solution overnight at 4°C. The samples were then incubated with a secondary antibody and stained with DAPI. The slides were observed under a confocal microscope (Carl Zeiss, LSM880).

Flow cytometry. The pri-miR-187, pri-miR-187/NIPBL, and scramble hESC-ECs were treated with StemPro Accutase Cell Dissociation Reagent (Gibco, A1110501) and incubated with APC-conjugated Mouse Anti–Human CD31 (WM59) (Thermo Fisher Scientific, 17-0319-42) in PBS at 4°C for 30 minutes in the dark. Collagenase II was used to dissociate mouse cardiac tissue at 37°C for 1 hour, followed by filtration through a 100 μm mesh to collect single-cell suspension. The suspension was treated with 1 mL of red blood cell lysis buffer at room temperature for 1 hour, centrifuged at 300g for 5 minutes to remove the supernatant, and subsequently incubated with FITC-conjugated Rat Anti–Mouse CD31 (WM59) (BD Pharmingen, 553372) and 647 Mouse Anti–Cardiac Troponin T (BD Pharmingen, 565744) in PBS at 4°C for 30 minutes in the dark. The cells were then sorted using a BD FACSCalibur (BD Biosciences) or Gallios (Beckman Coulter) flow cytometer. The resulting data were analyzed using FlowJo (BD Biosciences).

Mouse studies. The experimental procedures followed the Administrative Panel on Laboratory Animal Care protocol and the institutional guidelines by the Medical Ethics Committee at the Obstetrics and Gynecology Hospital of Fudan University. Rosa26 site-specific miR-187–knockin mice, also known as miR-187–KI mice, were created using the CRISPR/Cas9 system on a C57BL/6J background. These mice expressed a single copy of exogenous mmu-miR-187, controlled by the mouse Tek (Tie2) promoter.

To prepare for microinjection, capped mRNAs for Cas9 were generated using the mMESSAGE mMACHINE in vitro transcription kit (Invitrogen, AM1344) following the manufacturer’s instructions. The RNA’s integrity was verified by electrophoresis on a 1% agarose gel after denaturation using the loading buffer provided in the Invitrogen kit. Standard plasmid DNA preparation was used, followed by extraction with phenol/chloroform (Sangon, PD0419-1). The DNA was then diluted to 10 ng/μL with sterile microinjection TE buffer (0.1 mM EDTA, 10 mM Tris, pH 7.5; Solarbio, T1140) and stored at −80°C until the injection. We ensured RNase-free DNA by incubating it with in vitro–transcribed RNA at 37°C for 1 hour and analyzing the mix on a 1% agarose gel after denaturation using the loading buffer.

To generate Rosa26 (R26) site-specific miR-187–knockin mice, a donor plasmid containing a mouse miR-187 genomic fragment (5′-TCAGGCTACAACACAGGACCCGGGCGCTGCTCTGACCCCTCGTGTCTTGTGTTGCAGCCGG-3′) and flanking region controlled by a Tek promoter, a Tek enhancer, and a rabbit globin polyA signal sequence was constructed. The Cas9 mRNA and a single-guide RNA (sgRNA) targeting the R26 locus were generated, and the donor vector, Cas9 mRNA, and sgRNA (5′-GGCAGGCTTAAAGGCTAACC-3′) were co-microinjected into fertilized eggs from C57BL/6J mice, which were then transferred to pseudopregnant mice. The injection mixes contained 5 ng/μL DNA and 50 ng/μL of in vitro–transcribed Cas9 mRNA in microinjection TE buffer. Stable Mendelian transmission was confirmed, and RT-qPCR verified endothelial cell–specific expression of mmu-miR-187. The injection mixes were prepared before each injection by mixing of equal volumes of 10 ng/μL DNA solution and 100 ng/μL mRNA solution.

To confirm the site-specific insertions in the animals, we conducted 3 PCR tests: one for the junction at the 5′ end, one for the junction at the 3′ end, and one located internally within the transgene. The genomic DNA from their offspring was analyzed to confirm positive homologous recombination by PCR. After obtaining the heterozygous miR-187–KI mice, we established homozygous mice by backcrossing them with WT C57BL/6J mice and self-crossing the heterozygous mice. These lines were maintained by breeding of homozygous animals and exhibited normal fertility.

Echocardiographic studies. Mice were maintained on a heating platform to keep their body temperature at 36.5°C–37.5°C. The mice were anesthetized with 2% isoflurane and then kept under mild anesthesia during the echocardiographic procedure. Cardiac ultrasound was performed using the Vevo770 imaging system. Initially, the long and short axes of the mouse heart were visualized in B-mode, followed by analysis of the short axis in M-mode.

Open field test. Each mouse was placed in a corner of the open field apparatus (40 × 40 × 30 cm) with an illumination level of 100 lux. The number of entries into the central area (20 × 20 cm) and the duration spent there were recorded over a 10-minute period.

Light/dark transition test. The light/dark transition test was conducted using a cage (21 × 42 × 25 cm) divided into 2 equal compartments by a partition with a door. One compartment was brightly lit (390 lux), while the other remained dark (2 lux). Mice were placed in the dark compartment and allowed to freely move between the 2 compartments for 10 minutes with the door open. Transition frequency and time spent in each compartment were recorded using ImageLD software (Wayne Rasband, NIH).

Histological analysis. The hearts collected at E13.5 or P0 were fixed with 4% paraformaldehyde (pH 7.4; Sigma-Aldrich, P6148-1kg) for 30 or 50 minutes and then embedded in paraffin (Sangon, A606115). They were then sectioned at a thickness of 10 μm and subjected to hematoxylin and eosin (H&E) staining (Sangon, E607318-0200) for routine histological examination using a light microscope.

Transfection. The miRNA mimic (B02004) and miRNA inhibitor (B03004) were obtained from GenePharma and used for transfection experiments. To purify endothelial cells, endothelial cells derived from hESCs were purified by MACS at least once, reaching a minimum purity of 90%. Transfection of miRNA mimic, miRNA inhibitor, siRNA, or negative control was carried out using Lipofectamine RNAiMAX (Invitrogen, 13778075), while cotransfection of miR-187 mimic and NIPBL-expressing plasmids was performed using Lipofectamine 3000 (Invitrogen, L3000015). Cells were collected 48 hours after transfection.

Antibodies. Primary antibodies against the following proteins were used in this study: anti-SOX2 antibody (1:500 for immunofluorescence [IF]; Cell Signaling Technology, 3579T), anti-GAPDH antibody (1:5,000 for WB; Proteintech, 60004-1), anti–human CD31 (1:500 for IF; Abcam, ab9498), anti–mouse CD31 (1:500 for IF; BD Pharmingen, 557355), anti–von Willebrand Factor (1:500 for IF; Abcam, ab6994), FITC-conjugated mouse anti–human CD31 (WM59) (1:50 for FACS; BD Pharmingen, 557508), anti–human CD31 (PECAM-1) monoclonal antibody, APC (1:50 for FACS; eBioscience, 17-0311-82), wheat germ agglutinin, Alexa Fluor 488 conjugate (1:500 for IF; Invitrogen, W11261), anti-NIPBL antibody (1:1,000 for WB; 1:50 for CUT&Tag, Bethyl Laboratories, A301-779A-T), anti–histone H3 antibody (1:1,000 for WB; Cell Signaling Technology, 4499), anti–human phospho–histone H3 (Ser10) antibody (1:500 for IF; Cell Signaling Technology, 53348T), anti–mouse phospho–histone H3 antibody (1:500 for IF; Sigma-Aldrich, 06-570), anti–α-actinin antibody (1:500 for IF; Sigma-Aldrich, A7732), anti-WT1 antibody (1:500 for IF; Abcam, ab89901), anti-NFAT2 antibody (1:500 for IF; Abcam, ab25916), anti-H3K27Ac antibody (1:50 for CUT&Tag; Abcam, ab177178), and anti–normal rabbit IgG (1:50 for CUT&Tag; Cell Signaling Technology, 2729).

The secondary antibodies were goat anti-rabbit Alexa Fluor 488 antibody (1:500 for IF; Invitrogen, A-11008), goat anti-mouse Alexa Fluor 594 antibody (1:500 for IF; Invitrogen, A-11005), HRP-conjugated Affinipure Goat Anti-Rabbit Antibody (1:10,000 for WB; Proteintech, SA00001-4), and HRP-conjugated Affinipure Goat Anti-Mouse Antibody (1:10,000 for WB; Proteintech, SA00001-1).

Plasmids. The psiCHECK2-NIPBL-3′UTR luciferase reporter plasmid was created by amplifying a 449 bp fragment of the NIPBL 3′-UTR from human genomic DNA through PCR and cloning it into the XhoI and NotI sites of psiCHECK-2 (Promega). To generate the mutations plasmid corresponding to miR-187 binding sites, the plasmid of psiCHECK2-NIPBL-3′UTR-MUT was subjected to site-directed mutagenesis through PCR, and the resulting mutations were verified by DNA sequencing.

For the lentiviral vector pLJM1-pri-miR-187, a 586 bp human genomic DNA fragment, including pri-miR-187, was amplified by PCR and cloned into the NheI and EcoRI sites of pLJM1 (Addgene).

The NIPBL expression plasmids were constructed by cloning of the cDNA of NIPBL into pCDH-4HA.

Immunoblot analysis. In WB analysis, cells were washed with cold PBS and then lysed in cold Western lysis buffer (Beyotime, p0013) with a protease inhibitor cocktail (Roche, 04693132001). A standard procedure was used for the immunoblot analysis of total protein from the whole-cell lysate. GAPDH or H3 was used as an internal control to normalize the protein loading.

Luciferase reporter assay. The luciferase reporter plasmid psiCHECK2-NIPBL-3′-UTR or mutants were cotransfected into HEK293T or EA.hy926 cells seeded in 24-well plates along with 100 nM miR-187 mimic or miR-NC mimic and Lipofectamine 3000. After 36 hours, the cells were washed 3 times with cold PBS and lysed in a passive lysis buffer. Luciferase activity was measured using a Dual-Luciferase Assay System (Promega, E1960) on a GloMax-Multi Detection System plate reader (Promega).

miRNA pull-down assay. In the miRNA pull-down experiment, biotin-labeled double-stranded miR-187 mimic or miR-NC mimic was transfected into hESC-ECs with Lipofectamine RNAiMAX Transfection Reagent. After 24 hours, the cells were harvested, and RNA binding protein (RNP) complexes with the target mRNAs were pulled down by Dynabeads M-280 Streptavidin (Invitrogen, 11205D). To determine the binding specificity of miR-187 to NIPBL mRNA, RT-qPCR analyzed the target mRNAs, and the enrichment of the target mRNAs was calculated as follows: (NIPBL mRNA pulled down by miR-187 / NIPBL mRNA pulled down by miR-NC mimic) / (biotin–miR-187 input / biotin–miR-NC mimic input). The experiments were performed at least 3 times, with 3 replicates for each set.

Formation and culture of HFOs. The protocol of HFO formation was modified from previous publications (34). The hESCs were maintained on Matrigel Matrix (1:200; Corning, 354277) in mTeSR1 Plus medium. For HFO formation, hESCs were detached, and 3 × 104 cells per well were seeded in a U-shaped ultra-low-attachment 96-well plate (NEST, 701101) in mTeSR1 Plus medium. The plate was incubated to allow one aggregate per well to form overnight. On day 0, each aggregate was embedded in a Matrigel (Corning, 356231) droplet. Differentiation was initiated on day 0 by replacing the medium with RPMI 1640 medium containing B27 supplement without insulin (RB–) and supplemented with 7.5 μM CHIR-99021. After 24 hours, the medium was exchanged by RB−, and on day 1, RB– supplemented with 5 μM IWR-1 (Sigma-Aldrich, I0161) was added for 48 hours and then exchanged by RB− on day 3. From day 7 onward, aggregates were cultivated in RPMI 1640 medium containing B27 supplement with insulin (Gibco, 17504044) (RB+). Differentiation was completed on day 7. HFOs were analyzed between day 8 and day 10, and pictures were taken of the whole HFOs using a Castor X1 high-throughput cell analyzer (Countstar). Doxorubicin (500 nM) was administered during days –1 to 10 of HFOs.

Migration assay. The effect of miR-187 and NIPBL on hESC-EC migration was assessed using wound healing assays. A total of 1 × 105 cells were seeded in 6-well plates and allowed to culture for 24 hours. After 48 hours, transfection was performed using Lipofectamine 3000 with miR-187 mimic only, coexpressed miR-187/NIPBL, or scramble control. The cells were cultured until they reached confluence. Subsequently, scratches were created on the cell layers using a 1 mL pipette tip. The recovered area of the scratches was evaluated after 24 hours using an inverted light microscope.

Tube formation assay. hESC-ECs were seeded at 1 × 104 cells/cm2 density on a 24-well plate coated with 250 μL of Matrigel (Corning, 356231) in EGM-2 medium. The plate was then incubated for 24 hours at 37°C in a 5% CO2 atmosphere. After incubation, the medium was removed, and the plates were washed with PBS. The formation of capillary-like structures was observed using an inverted light microscope. Tube formation was quantified using ImageJ 1.52a software (Wayne Rasband, NIH) and the Angiogenesis Analyzer plug-in (Gilles Carpentier, Université Paris Est Créteil Val de Marne, Créteil, France).

Zebrafish studies. In a zebrafish study, exogenous dre-miR-187-3p mimic or negative control (all at 20 pM) was microinjected into fertilized cmlc2-DsRed (labeling CM nucleus) or cmlc2-EGFP (labeling CM membrane) zebrafish embryos. Zebrafish cardiac morphology was measured with confocal microscopy 72 hours after fertilization.

Statistics. Data are presented as mean values with corresponding standard deviations (SDs). Tukey’s multiple-comparison test was used for Figure 3, A, C–H, M, and N, Figure 4, I–N, Figure 5, C–F, Supplemental Figure 3, C, E, F, and L–N, Supplemental Figure 5, A and C–F, Supplemental Figure 6E, Supplemental Figure 8, C–F, Supplemental Figure 10, A, C, and D, and Supplemental Figure 13D. Other statistical significance of the differences between groups was determined using 2-sided Student’s t tests, and P values are reported. Differences in phenotype frequencies (Supplemental Figure 3B) between the KI/KI, KI/+, and +/+ mice were evaluated using Pearson’s χ2 test. The significance level is denoted by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Study approval. All procedures using mice for the current study were approved by the Institute of Developmental Biology and Molecular Medicine of Fudan University. All procedures using human specimens for the current study were approved by the Institutional Review Board of Fudan University. All experiments involving human tissue samples were performed following the Declaration of Helsinki. All experiments involving human tissue samples and animals were conducted with approval from the Medical Ethics Committee at the Obstetrics and Gynecology Hospital of Fudan University.

Data availability. All data are available upon reasonable request. The datasets generated during this study were uploaded to the Gene Expression Omnibus database under the following accession codes: GSE275849, GSE275950, and GSE275951 for RNA-Seq; GSE276221 and GSE276222 for ATAC-seq; GSE275850 for CUT&Tag-seq. Supporting data values are available in the Supporting Data Values file.

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