Fetal hepatocytes protect the HSPC genome via fetuin-A

Human tissue acquisition

Human fetal liver tissues and cord blood samples were obtained from Tongji Hospital, Tongji University School of Medicine, Shanghai, China, with written informed consent from the parents and approval from the Medical Ethics Committee (k-w-2010-010) of Tongji Hospital. Fetal developmental age was estimated from measurements of crown–rump length and compared with a standard growth chart⁴². Plasma from infant patients with leukaemia and benign patients was obtained from Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China, with written informed consent from guardians of the patients and approval from the Medical Ethics Committee (SCMCIRB-K2024163-1) of the Shanghai Children’s Medical Center.

Mice

B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J, B6.Cg-Tg(Alb-cre)21Mgn/J, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, C57BL/6J (B6-Ly5.2) and C57BL/6JGpt-Fetua-knockout (cas9) mice were maintained and bred in a pathogen-free facility in ventilated cages, a maximum of six mice per cage, on a 12-h day–night cycle, at 20–26 °C and 30–70% humidity, in compliance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. For embryo collection, 8–10-week-old male and female mice were mated at night and then separated the next morning; the time of separation was considered E0.5. For colony-forming cell assays, whole-genome sequencing and leukaemic models, 3-week-old mice were used. Male and female mice were used in all experiments. Mice were placed into groups depending on their gestational days and genotypes; when possible, mice were randomized and the group allocation was blinded. No sample size calculation was performed. All animal experiments were approved by the Institutional Animal Care and Use Committees of Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Isolation of human HSPCs

Liver tissues were processed immediately after isolation. The tissues were dissected into single-cell suspensions, and mononuclear cells were then separated using Ficoll density gradient centrifugation. Lineage-positive cells were depleted using the MagniSort Human Haematopoietic Lineage Depletion Kit (8804-6836-74, Thermo Fisher). Lineage-negative (Lin^−) cells were incubated with a combination of biotin-labelled lineage antibodies (to CD2, CD3, CD10, CD11b, CD14, CD16, CD19, CD56, CD123 and CD235a; 8804-6836-74, Invitrogen) and FITC-labelled CD34 (581; 555821, BD). After 15 min at 4 °C, the cells were washed with PBS, suspended in magnetic bead selection buffer (MACS; PBS, 2 mM EDTA and 0.5% BSA), and then incubated with streptavidin–phycoerythrin (12-4317-87, Thermo Fisher). After 10 min at room temperature, the cells were washed and suspended in IMDM (12440053, Thermo Fisher) supplemented with 1% BSA. Lin^− and CD34⁺ HSPCs were subjected to flow cytometry on an Aria III flow cytometer (BD), and the data were collected using BD FACSDiva (V8.0.3). The HSPCs were cultured in StemSpan medium (09650, Stem Cell) supplemented with 10 ng ml^−1 IL-6 (200-06, PeproTech), 10 ng ml^−1 IL-3 (200-03, PeproTech), 10 ng ml^−1 stem cell factor (SCF; 300-07, PeproTech) and 10 ng ml^−1 Flt3 (300-19, PeproTech) at a concentration of 1 × 10⁵ per millilitre for further experiments.

Isolation of mouse HSPCs

Pregnant mice were anaesthetized and euthanized by cervical dislocation. Placentas, fetal livers or bone tissues were then dissected into single-cell suspensions. Subsequently, the cells were incubated with biotin-labelled monoclonal antibodies targeting haematopoietic lineage markers (including B220, CD3, Gr-1 and Ter119; 88-7774-75, Thermo Fisher). After incubation, the cells were washed with MACS buffer and stained with streptavidin-conjugated magnetic beads (558451, BD). Following a 20-min incubation at 4 °C, the cells were washed again and resuspended in MACS buffer. Lineage-positive cells were depleted using a magnetic system. The Lin^− cells were then incubated with biotin-labelled lineage markers (88-7774-75, Thermo Fisher), phycoerythrin–Cy7-labelled Sca-1 (D7; 25-5981-82, Thermo Fisher), APC-labelled Kit antibodies (2B8; 17-1171-82, Thermo Fisher), phycoerythrin-labelled CD150 (mShad150; 12-1502-82, Thermo Fisher) and FITC-labelled CD48 antibodies (HM48-1; 11-0481-82, Thermo Fisher). After a 15-min incubation at 4 °C, the cells were washed with PBS and resuspended in MACS buffer. Streptavidin–phycoerythrin (12-4317-87, Thermo Fisher) or streptavidin–APC–Cy7 (405208, BioLegend) was added to the cells, which were then incubated for 10 min at room temperature. The cells were then washed and resuspended in IMDM supplemented with 1% BSA. LSK cells, lineage-negative, Sca-l-positive, Kit-positive, CD150-positive and CD48-negative cells (LT-HSCs), lineage-negative, Sca-l-positive, Kit-positive, CD150-negative and CD48-negative cells (ST-HSCs), and lineage-negative, Sca-l-positive, Kit-positive, CD150-negative and CD48-positive cells (MPPs) were flow-sorted according the gating strategy in Supplementary Fig. 1 in the Aria III flow cytometer (BD), and the data were collected using BD FACSDiva (v8.0.3). The HSPCs were cultured in StemSpan medium (09650, Stem Cell) supplemented with 10 ng ml^−1 IL-6 (216-16, PeproTech), 10 ng ml^−1 IL-3 (213-13, PeproTech) and 10 ng ml^−1 SCF (250-03, PeproTech) at a concentration of 1 × 10⁵ per millilitre for further experiments.

Isolation and culture of mouse fetal hepatocytes

To obtain Alb-Cre;ROSA26-LSL-tdTomato fetuses, we cross-mated B6.Cg-Tg(Alb-cre)21Mgn/J and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice. Fetal livers were then removed at E12.5 or E16.5 and digested with 0.6 mg ml^−1 collagenase IV (17104019, Thermo Fisher) in Hank’s balanced salt solution for 20 min at 37 °C. The digestion reaction was halted using cold PBS, and the mixture was subsequently centrifuged at 500 rpm for 5 min. Magnetic cell sorting (558451, BD) was used to remove blood cells expressing Ter119, B220, CD3, Gr-1 and Mac-1. Tomato-positive hepatocytes were flow-sorted using an MoFlo Astrios flow cytometer, and the data were collected using Summit (v6.3.1.16945; Beckman Coulter). The isolated hepatocytes were precultured in StemSpan medium (09650, Stem Cell) supplemented with hepatocyte growth supplement (1:100; 5201, ScienCell), 2 ng ml^−1 IL-6 and 5% FBS (F2442, Sigma; referred to as SHIF) for 4 h. After removing the medium and non-adherent cells, the adherent hepatocytes were cultured in the same medium without FBS (referred to as SHI) for 20 h. The supernatant from the cultured fetal hepatocytes (referred to as conditioned SHI or co-SHI) was collected for further experiments.

Comet assay

The HSPCs at different development stages and HSPCs cultured with or without FetuA (100 μg ml^−1; 10318-H08H, SinoBiological) or ML216 (25 mΜ; S0469, Selleck) were treated with 20 μM etoposide (341205, Merck Millipore) for 30 min and then collected for the comet assay. A CometAssay kit from TREVIGEN (4250-050-k, R&D) was utilized to assess DNA damage. The alkaline method was utilized using the following steps: (1) 5,000 cells were mixed with 50 µl of melted LMAgarose (4250-050-02, R&D) at 37 °C and then pipetted onto a CometSlide; (2) the cells were then gelled for 3–5 min at 4 °C in the dark, followed by lysis with 4 °C lysis solution for 1 h; (3) the CometSlide was immersed in an alkaline unwinding solution (200 mM NaOH and 1 mM EDTA, pH > 13) for 1 h at 4 °C in the dark, and electrophoresis was conducted in the same solution at 20 V and 300 mA for 30 min at 4 °C); (4) the slides were washed twice with ddH₂O for 5 min each, followed by washing with 70% ethanol for 5 min, and the slides were then dried at room temperature overnight; and (5) the dried agarose was stained with SYBR Green I (A25742, Thermo Fisher) nucleic acid gel stain for 30 min. Images were acquired with Las X (v4.7) on a Leica TCS Stellaris8 STED Microscope at ×20 resolution and analysed using OpenComet software (v1.3.1).

Immunofluorescence staining of cells

Human HSPCs treated with 80 µM etoposide (341205, Merck Millipore) for 30 min; mouse HSPCs treated with 20 µM etoposide for 30 min, 500 J m^−2 ultraviolet radiation B (UVB) irradiation; human HSPCs cultured with or without FetuA (100 μg ml^−1; 10318-H08H, SinoBiological) for 2 h and then treated with 80 µM etoposide; mouse HSPCs cultured with co-SHI or hepatocytes for 2 h and then treated with 20 µM etoposide; and mouse HSPCs pre-treated with or without TLR4 antibody (1:50; 53-9041-80, Thermo Fisher) at 4 °C for 30 min and then cultured with or without FetuA (100 μg ml^−1; 50093-M08H, SinoBiological) for 2 h and thereafter treated with 20 µM etoposide were collected. These cells were then spun onto slides and fixed in 4% PFA for 10 min. After treatment with PBS containing 2% serum, 1% BSA and 0.2% Triton X-100, the cells were directly stained with primary antibodies overnight at 4 °C. Subsequently, the sections were stained with secondary antibodies for 1 h at room temperature. Phosphor-histone H2AX (Ser13; 20E3) rabbit monoclonal antibody (1:400; 9718S) was acquired from Cell Signaling Technology (CST), and TLR4 antibody (1:200; ab13556), mouse FetuA antibody (EPR17839-163; 1:100; ab187051) and p-RPA (phospho S33; 1:200; ab211877) were obtained from Abcam. Human FetuA antibody (1F6B9; 1:100; 66094-1-Ig) was obtained from Proteintech. The MYD88 (E11; 1:400; sc-74532) antibody was acquired from Santa Cruz. Images were acquired with ZEN (v2.3) on a Zeiss 880 Microscope at ×20 resolution or ×63 resolution, and analysed using ImageJ (v1.52p) and HALO (v3.6.4134).

Immunofluorescence staining of whole-mount tissues

Fetal livers and placentas were fixed in 4% PFA for 30 min, washed with PBS for 2–3 h, and stained with primary antibodies (diluted in PBS containing 1% BSA, 2% FCS and 0.5% Triton X-100) for 1–3 days. The tissues were then incubated with secondary antibodies for 2 h. Anti-mouse CD117 (Kit, ACK2; 1:50; 14-1172-85) was acquired from Thermo Fisher, and phospho-histone H2AX (Ser139; 20E3; 1:400; 9718S) rabbit monoclonal antibody was obtained from CST. Images were acquired with Las X (v4.7) on a Leica TCS Stellaris8 STED Microscope at ×20 resolution, and analysed using Image J (v1.52p) and Imaris (v9.0.1).

Immunofluorescence staining of tissue sections

Human or mouse placenta, fetal liver and bone tissues were fixed in 4% PFA for 30–60 min at room temperature (placenta and fetal liver tissues) or 5 h at 4 °C (bone tissues), dehydrated in 15% and 30% sucrose, and embedded in optimal cutting temperature compound at −20 °C. The tissues were then sectioned (20–25 μm) using a cryostat. The tissue sections were stained with primary antibodies for 6–12 h at 4 °C in PBS containing 1% BSA, 2% FCS and 0.5% Triton X-100. The sections were incubated with secondary antibodies for 1 h at room temperature. CD48–FITC (HM48-1; 1:100, 11-0481-82) was acquired from Thermo Fisher. APC-anti-lineage (Ter119 (1:400, 116212), Gr-1 (1:400, 108412), Mac-1 (1:400, 101212), B220 (1:400, 103212), CD3 (1:100, 100236), CD150-BV421 (SLAM; 1:100, 115925) and CD41-APC (MWReg30; 1:400, 133913)) were acquired from BioLegend. Kit goat monoclonal antibody (Gln25–Thr519; Ala207Glu; 1;400, AF1356) and anti-human serum albumin antibody (MAB1455; 1:200, 188835) were acquired from R&D. E-cadherin rabbit monoclonal antibody (24E10; 1:200, 3195T) and phospho-histone H2AX (Ser139; 20E3; 1:400, 9718S) rabbit monoclonal antibodies were acquired from CST. Laminin monoclonal antibody (1:200, ab11575), mouse FetuA antibody (EPR17839-163; 1:400, ab187051) and CD34 antibody (EP373Y; 1:100, ab81289) were acquired from Abcam. CD45–FITC (104; 1:100, MCD45201) was acquired from Invitrogen. Nestin antibody (1:50, AN205-1) was acquired from Beyotime. E-cadherin antibody (DECAM-1; 1:200, sc-59778) was acquired from Santa Cruz. CD144 antibody (1:200, 550548) and Sca-1 antibody (D7; 1:200, 557403) were acquired from BD. Human FetuA antibody (1F6B9; 1:200, 66094-1-Ig) was acquired from Proteintech. Images were acquired with ZEN (v2.3) on a Zeiss 880 Microscope at ×20 resolution or ×63 resolution, and analysed using ImageJ (v1.52p), Imaris (v9.0.1) and HALO (v3.6.4134).

Colony-forming cell assay

The assay was performed in a semi-solid methycellulose medium (03434, Stem Cell Technologies) following the technical manual. In brief, the sorted HSPCs were plated in methycellulose in a 35-mm dish (200 cells per dish). Cultures were incubated at 37 °C in a humidified incubator (more than 95%) with 5% CO₂ in the air. The colonies were scored under a microscope 10–12 days post-plating. Replating was performed by pooling total cells from primary cultures and inoculating 10⁴ cells into fresh methycellulose medium.

Metaphase chromosome preparation and FISH

Bone marrow Lin^− cells were incubated with 0.05 μg ml^−1 colcemid for 1 h at 37 °C and then centrifuged at 400g for 10 min. The cells were suspended in 1 ml of hypotonic solution (0.075 M KCl) for 30 min at 37 °C, and the reaction was stopped by the addition of freshly prepared fixative solution (3:1 methanol:glacial acetic acid). The cells were then subjected to three rounds of fixative changes. After that, the cells were dropped onto slides and allowed to dry at room temperature. Two-colour FISH was performed using whole-chromosome probes for mouse chromosome 4 (FITC; D-1404-050-FI, MetaSysterms) and chromosome 6 (Texas red; D-1406-050-OR, MetaSysterms), and counterstaining was performed with DAPI-Antifade solution. Images were acquired with Las X (v4.7) on a Leica TCS Stellaris8 STED Microscope at ×20 resolution and analysed using Image J (v1.52p).

Mouse models of leukaemia

Three-week-old mice were intraperitoneally injected with 80 mg kg^−1 N-ethyl-N-nitrosourea (ENU; N3385, Sigma) four times, which was administered twice a week, and the mice were monitored by daily observation of leukaemic symptoms and signs including fired hair, white toes, swollen lymph nodes and loss of body weight, and weekly measurement of leukaemia-like cells by Gimsa staining of peripheral blood smears. Once these appeared, the animals were killed, and the disease of leukaemia was determined by exhibition of enlarged spleens and lymph nodes, increased proportions of immature Lin^− cells in the bone marrow and leukaemic cell infiltration in spleens and bone marrow shown by haematoxylin and eosin staining of the tissue sections.

Western blot

After treatment with or without FetuA (100 μg ml^−1; 50093-M08H, SinoBiological) for 2 h, mouse HSPCs (LSK) were lysed and blotted with bZIP antibody and their phosphorylated forms. After treatment with or without FetuA (100 μg ml^−1; 50093-M08H, SinoBiological) and the bZIP inhibitor SR11032 (2 mM; HY-15870, MedChemExpress) for 6 h, mouse Lin^– haematopoietic cells were lysed and blotted with BLM antibody. The cells were lysed using SDS lysis buffer. The lysates were then separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Western blotting was carried out using the following primary antibodies: FetuA (1:2,000), JunB (1:1,000), phosphorylated JunB (p-JunB; 1:1,000), Jun (1:1,000), p-Jun (1:1,000), Fosl1 (1:10,000), p-Fosl1 (1:1,000), BLM (1:500) and laminB1 (1:2,000). The membranes were incubated overnight at 4 °C with primary antibodies. After rinsing to remove any unbound primary antibody, the membranes were exposed to a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The secondary antibody was detected using chemiluminescence (WBKLS0500, Merck Millipore). The following primary antibodies were used: anti-p-JunB (Thr102/Thr104; 8053S) and anti-p-Fosl1 (S265; 3880S) from CST and anti-JunB (EPR6518; ab128878), anti-Fosl1 (ab232745), anti-p-Jun (phospho S63; ab32385), anti-Jun (EP693Y; ab40766), anti-laminB1 (EPR8985; ab133741) and anti-FetuA (EPR17839-163; ab187051) from Abcam, and anti-BLM (B-4; sc-365753) from Santa Cruz. The intensity of bands was measured using ImageJ (v1.52p). For gel source data, see Supplementary Fig. 2.

Mass spectrometry analysis

SHI and co-SHI media, E12.5 and E16.5 fetal liver tissues and bone marrow plasma samples from infants were collected for data-independent acquisition tandem mass spectrometry (DIA MS/MS) analysis. The medium samples were treated with 2% SDS buffer containing 50 mM dithiothreitol for 20 min at room temperature and then boiled at 100 °C for 5 min. The protein samples were alkylated in the dark at room temperature for 1 h by adding 200 mM iodoacetamide. To precipitate the proteins, a 5× volume of pre-cooled acetone was used overnight at −20 °C. The tissue samples were diluted with 50 mM NH₄HCO₃ and centrifuged three times at 20 °C and 14,000g using YM-10 filter units. The protein lysates were reduced for 1 h at room temperature with a final concentration of 10 mM dithiothreitol and then alkylated for 1 h in the dark at room temperature with a final concentration of 55 mM iodoacetamide. The protein mixtures were exchanged with 50 mM NH₄HCO₃ by centrifugation at 20 °C and 14,000g three times. The protein precipitates were digested overnight at 37 °C at a protein-to-enzyme ratio of 50:1 with trypsin. Tryptic peptides were collected by centrifugation at 20 °C and 14,000g for 20 min. The peptides were then treated with 1% trifluoroacetic acid, purified using C18 Ziptips and eluted with 0.1% trifluoroacetic acid in 50–70% acetonitrile. The eluted peptides were dried using a SpeedVac (Thermo Savant) and resuspended in 1% formic acid and 5% acetonitrile. Before analysis, indexed retention time (iRT) peptides (Biognosys) were spiked into the samples following the manufacturer’s instructions. The pooled digestates were dried using a SpeedVac (Thermo Savant) and resuspended in 5% ACN in 0.05 M ammonium formate. The digested peptides were fractionated using high-pH reversed-phase separation on a Dionex ultra-high-performance liquid chromatography (Thermo Scientific) with a 2.1 × 150 mm ethylene-bridged hybrid (BEH) C18 3-μm column at 40 °C, with a flow rate of 0.2 ml min^−1 and a 60-min ACN gradient (5–30%) in 5 mM ammonium formate (pH 10). Fractions were collected at 1-min intervals and pooled at various intervals, resulting in up to 12 fractions. The samples were dried and resuspended in 1% formic acid and 5% acetonitrile. Data-dependent acquisition (DDA) analysis was conducted on an Orbitrap Fusion LUMOS mass spectrometer (Thermo Fisher Scientific) connected to an Easy-nLC 1200 via an Easy Spray (Thermo Fisher Scientific). The peptide mixtures were loaded onto a self-packed analytical PicoFrit column (75 μm × 40 cm) with an integrated spray tip (New Objective) packed with ReproSil-Pur 120A C18-AQ 1.9 μm (Dr. Maisch GmbH). The peptides were separated using a 120-min linear gradient from 95% solvent A (0.1% formic acid, 2% acetonitrile and 98% water) to 28% solvent B (0.1% formic acid and 80% acetonitrile) at a flow rate of 250 nl min^−1 at 50 °C. The mass spectrometer was operated in positive-ion mode and used the data-dependent mode with a specialized cycle time (3S) to automatically switch between MS and MS/MS scans. A full MS scan from 350 to 1,500 m/z was acquired at a resolution of R = 120,000 (defined at m/z = 400). MS/MS scans were performed at a resolution of 30,000, with an isolation window of 4 Da and higher-energy collisional dissociation fragmentation with a collision energy of 30 ± 5%. Dynamic exclusion was set to 30 s. Sequences were identified using the mouse UniProt fasta database (53,099 entries, downloaded on 4 November 2018) with default parameters. The digestion enzyme used was a specific trypsin enzyme with two missed specialized cleavages. Carbamidomethyl of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. The iRTs derived from median iRTs across all DDA runs were calculated. Fragment ions for the targeted data analysis were selected from 300 to 1,800 m/z, with a minimal relative intensity set to more than 5% and a fragment ion number greater than 3. The false discovery rate (FDR) was set to 1% for protein and peptide spectrum matches. Protein inference was performed using the ID Picker algorithm integrated within the Spectronaut software. DIA MS/MS acquisition was performed using the same liquid chromatography-MS systems and liquid chromatography linear gradient method as DDA. For MS/MS acquisition, the DIA method was set with 50 variable isolation windows based on the full-width at half-maximum and constructed using the respective DDA data. The full scan was set at a resolution of 1,200,000 over a m/z range of 350–1,500, followed by DIA scans at a resolution of 30,000. The collision energy (CE), auto gate control (AGC) and maximal injection time were set to 30 ± 5%, 1 × 10⁶ and 54 ms, respectively. The DIA raw files were analysed using Spectronaut X (Biognosys). The retention time prediction type was set to dynamic iRT, and a correction factor was applied for window 1. Interference correction at the MS2 level was enabled. Systematic variance was normalized using a local normalization strategy. The FDR was estimated using the mProphet approach and set to 1% at the peptide precursor and protein levels. Protein intensity was calculated by summing the intensities of their respective peptides, which were measured using the peak areas of their fragment ions in MS2 and multiplied by a factor based on the total sample volume of each sample. All the results were filtered with a Q value cut-off of 0.01 (corresponding to a 1% FDR).

Intraplacental injection of FetuA

The pregnant mice were anaesthetized using 3.5% chloral hydrate and then secured onto a heating pad with all four legs immobilized. The abdominal surface was shaved and disinfected with 75% alcohol. A longitudinal incision measuring 1–1.5 cm in length was made on the abdominal skin, and the peritoneum was cut. Cotton gauze was placed around the incision. One uterine horn was carefully exposed and pulled out using blunt forceps onto gauze soaked in PBS. The uterus was held in place with blunt forceps, and recombinant FetuA (10 μg per 15 μl each) was injected into the placenta. After the injection, the uterus was carefully returned to the abdomen, ensuring that it was positioned exactly as before. The peritoneum was closed with a haemostat. Two hours later, 5 mg kg^−1 etoposide was administered through intraperitoneal injection. The fetuses were harvested after 1 h and fixed for immunofluorescence assays.

ATAC-seq library preparation and sequencing

ATAC-seq libraries were prepared as previously described⁴³. In brief, HSPCs were lysed using lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂ and 0.1% IGEPAL CA-630 for 10 min before being spun at 4 °C to obtain nuclear preparations. The supernatant was discarded, and the nuclei were then incubated with Tn5 transposome and tagmentation buffer at 37 °C for 30 min (Vazyme). The resulting tagmentation products were purified and amplified using PCR. PCR amplification involved ten cycles under the following conditions: 72 °C for 5 min; 98 °C for 30 s; thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min; and 72 °C for 5 min. The libraries were purified using a PCR purification kit (28004, Qiagen), and the fragments were enriched using 0.5× and 1.0× VAHTS DNA Clean Beads (N412-01, Vazyme) after amplification.

ATAC-seq data processing

All reads were aligned to the mm10 genome using the Burrows-Wheeler Aligner (BWA-MEM) after trimming the adapter sequences with Trim_Galore (v0.6.7). Low-quality reads were filtered out, whereas PCR duplicates and reads mapped to the mitochondria or the Y chromosome were discarded. The remaining reads on the left were shifted (+4/−5) to correct Tn5 enzyme insertions based on the read strands. Peak calling was performed using MACS2 (v2.2.6) with the following options: -f BAM, -g mm, –nomodel, –shift -100 and –extsize 200. The samples were normalized using the bamCoverage function from deepTools (v3.5.1) to visualize the signal in IGV (v2.7.0).

Quality control of ATAC-seq data was conducted, and correlation analysis was performed using deepTools (v3.5.1). The fragment distribution was generated using ATAC-seq QC (v1.14.4). The peak atlas was obtained by expanding the peak summit by ±500 bp, and differential peaks were identified using DESeq2 (v1.26.0). DNA-binding factor motifs were analysed by determining the motifs in the differential peaks using HOMER (v4.11). ATAC signals were visualized as a heatmap using the complexHeatmap package (v2.2.0).

RNA-seq and analysis

Total RNA was extracted from cultured or non-cultured HSPCs using Tri Pure Isolation Reagent (11667157001) from Roche following the manufacturer’s instructions. The quality of the RNA was assessed using the Fragment Analyser platform. High-quality samples were chosen for library construction using the Illumina TruSeq RNA Prep Kit (20015949). The libraries were subsequently sequenced on the Illumina HiSeq4000 platform, generating 2 × 150 bp paired-end reads. To process the raw data, Trim Galore (v0.6.7) was used with the following parameters: ‘–quality 20 –fastqc –length 20 –stringency 1’. The resulting clean reads were then aligned to the mouse reference genome (mm10) using hisat2 (v2.2.1). GENCODE annotations (gencode.vM25.annotation.gtf; downloaded in April 2021) and HTSeq-count (v0.13.5) were used to assign the aligned reads to genes. Subsequently, the counts were normalized to fragments per kilobase of transcript per million mapped reads (FPKM), and log(FPKM + 1) was utilized to analyse the overall similarity or dissimilarity between the samples.

Cut&Tag library preparation and sequencing

The Hyperactive Universal Cut&Tag Assay Kit for Illumina Pro (TD904, Vazyme) was used in this study. In summary, 1 × 10⁵ LSK cells treated with or without FetuA were collected and washed in 500 µl of wash buffer. The cells were then resuspended in 100 µl of wash buffer. Subsequently, 10 µl of concanavalin A-coated magnetic beads were activated and added to 1 × 10⁵ cells. The cells were incubated at room temperature for 10 min, after which the supernatant was removed. The resulting bead-bound cells were resuspended in 50 µl of antibody buffer. Next, 1 µl of Jun rabbit monoclonal antibody (60A8; 9165T, CST), JunB rabbit monoclonal antibody (C37F9; 3753S, CST) or Fosl1 mouse monoclonal antibody (C-12; sc-28310, Santa Cruz) was added and incubated with the bead-bound cells overnight at 4 °C with rotation. The supernatant was then removed, and the bead-bound cells were resuspended in 50 µl of dig-wash buffer containing goat anti-rabbit IgG antibody (Ab207-01-AA, Vazyme) or goat anti-mouse IgG antibody (Ab208-01-AA, Vazyme; diluted 1:100). This mixture was incubated at room temperature for 1 h. The bead-bound cells were washed three times with 200 µl of dig-wash buffer to remove any unbound antibodies. Next, 2 µl of the pA-Tn5 adapter complex was diluted in 98 µl of dig-300 buffer and mixed with the bead-bound cells. The mixture was subjected to rotation at room temperature for 1 h. The bead-bound cells were washed three times with 200 µl of dig-300 buffer to eliminate any unbound pA-Tn5 protein. The cells were then resuspended in 50 µl of tagmentation buffer and incubated at 37 °C for 1 h. To terminate the tagmentation reaction, 2 µl of SDS was added to the cells and incubated for an additional 10 min at 55 °C. The tagmentation products were purified using DNA Extract Beads Pro and eluted in 15 µl of nuclease-free water. For generation of the sequencing libraries, the DNA tagments were mixed with a universal i5 primer and a unique i7 primer and amplified using 2× Cut &Tag amplification mix. The resulting PCR products were purified using VAHTS DNA lean beads (N411, Vazym), and subsequently analysed using an Agilent 2100 Bioanalyzer and Illumina Novaseq 6000.

Cut&Tag data processing

Cut&Tag reads were aligned to the mm10 genome with Bowtie2 (v2.3.5.1) using the following parameters: –end-to-end –very-sensitive –no-mixed –no-discordant –phred33 -I 10 -X 700. Duplicate reads were removed with Picard (v2.25.5). The track files were made with the bamCoverage command from deepTools (v3.5.1). Cut&Tag peaks were called using MACS2 (v2.2.6). The distribution of Cut&Tag peaks was annotated with the R package ChIPseeker (v1.22.1).

R-loop staining

HSPCs from E12.5 placenta, E12.5 fetal liver, E16.5 fetal liver or E12.5 FL-HSPCs with or without FetuA (100 μg ml^−1; 50093-M08H, SinoBiological) or ML216 (25 mΜ; S0469, Selleck) treatment for 2 h were collected. The cells were spun onto slides, fixed in 4% PFA for 10 min and washed three times with PBS. After permeabilization with PBS containing 0.3% Triton X-100 for 10 min at room temperature, the cells were washed three times with PBS and then blocked with PBS containing 2% serum, 1% BSA and 0.2% Triton X-100 for 1 h at 37 °C. Immunofluorescence experiments with the dRNH1 protein were performed as previously described³³. In brief, the cells were incubated with 30 μl dRNH1 (0.24 mg ml^−1) for 1 h at 37 °C, followed by three washes with PBS. The cells were then incubated with 1 μg ml^−1 DAPI for 10 min. For immunofluorescence experiments with the GST-His6-2×HBD protein³², the cells were incubated with 30 μl GST-His6-2×HBD (2 μg ml^−1) overnight at 4 °C. After three washes with PBS, the cells were stained with an anti-HisTag monoclonal antibody (AMC0149; 1:400; AE003, ABclonal) for 1 h at room temperature, followed by three washes with PBS. The cells were then stained with a rabbit anti-mouse IgG antibody (1:400; SPA231, Solarbio) for 1 h at room temperature. After three washes with PBS, the cells were incubated with 1 μg ml^−1 DAPI for 10 min. Images were acquired using a Zeiss 880 microscope, and the signal intensity was measured using HALO (v3.6.4134).

R-loop Cut&Tag library preparation and sequencing

The R-loop Cut&Tag library was prepared following protocols previously described³² with minor modifications. For this experiment, the Hyperactive Universal Cut&Tag Assay Kit for Illumina (TD903, Vazyme) was utilized. In brief, 1 × 10⁵ cells were gently pipetted and washed twice in 500 µl of wash buffer. Then, 10 µl of concanavalin A-coated magnetic beads was activated and added to the 1 × 10⁵ cells, followed by incubation at room temperature for 10 min. The supernatant was then removed, and the bead-bound cells were resuspended in 90 µl of antibody buffer. Subsequently, 10 µl of recombinant GST-His6-2×HBD (0.2 mg ml^−1) protein was added and the mixture was incubated with the bead-bound cells overnight at 4 °C with rotation. After two washes with dig-wash buffer, the samples were incubated with an anti-HisTag monoclonal antibody (AMC0149; 1:400; AE003, ABclonal) for 1 h at room temperature, followed by incubation with a rabbit anti-mouse IgG antibody (final concentration, 10 µg ml^−1; SPA231, Solarbio) for 1 h at room temperature. Unbound antibodies were removed by three brief washes with 200 µl of dig-wash buffer. To facilitate tagmentation, 2 µl of a pA-Tn5 adapter complex was diluted in 98 µl of dig-300 buffer and mixed with bead-bound cells, which were then rotated at room temperature for 1 h. After three washes in 200 µl of dig-300 buffer to remove unbound pA-Tn5 protein, the cells were resuspended in 50 µl of tagmentation buffer and incubated at 37 °C for 1 h. The tagmentation reaction was stopped by adding 1.8 µl of 0.5 M ethylenediaminetetraacetic acid, 0.6 µl of 10% SDS, 5 µl of nuclease-free water and 1 µl of proteinase K (20 mg ml^−1), and further incubated at 55 °C for 60 min. Following purification with 1× DNA clean beads (Vazyme Biotech), the resulting tagmentation products were eluted in 10 µl of nuclease-free water. For the strand displacement reaction, the eluent was mixed with 10 U of Bst 2.0 WarmStart DNA polymerase (M0538, NEB) in 1× Q5 polymerase reaction buffer and incubated at 65 °C for 30 min. The reaction was then halted by incubation at 80 °C for 20 min. To generate the sequencing libraries, the mixture was combined with a universal i5 primer and a uniquely barcoded i7 primer and subsequently amplified using Q5 high-fidelity master mix (M0491, NEB). The libraries were size selected with 0.56–0.85× DNA clean beads and subjected to analysis using an Agilent 2100 Bioanalyzer and Illumina PE150 sequencing.

R-loop Cut&Tag data processing

The Cut&Tag data were processed as previously described³². In summary, Cut&Tag reads were aligned to the mm10 genome using Bowtie2 (v2.3.5.1), allowing for uniquely mapped reads with up to two mismatches. The aligned reads were normalized to the total number of reads (reads per million). Subsequently, track files were generated using the bamCoverage command from deepTools (v3.5.1). Cut&Tag peaks were called using MACS2 (v2.2.6), and the distribution of Cut&Tag peaks was annotated using the R package ChIPseeker (v1.22.1). The average coverage was used to create metaplots within the indicated windows. Gene Ontology enrichment analysis was performed using clusterProfiler (v3.14.3), and circus-plots were generated using circlise (v0.4.8).

Whole-genome sequencing of mouse HSPCs

Genomic DNA was extracted using the QIAamp DNA Mini Kit (51304, Qiagen) following the manufacturer’s protocol. Whole-genome sequencing was conducted as previously described⁴⁴. In brief, short-insert 350-bp genomic libraries were constructed following Illumina library protocols, and sequencing was performed on an Illumina NovaSeq 6000 platform using 150-base paired-end reads, achieving an average coverage of 30×. The sequence data were mapped to the mouse genome reference mm10 using the BWA-MEM algorithm. Unmapped reads and PCR-derived duplicates were excluded from the analysis. Insertions and deletions (indels) and structural variants were called using the Pindel and BreakDancer algorithms, respectively, as described elsewhere^45,46. The group treated with saline was used as germline control.

Cell cycle analysis of mouse HSPCs by combining Hoechst and pyronin Y

The cells were harvested from E12.5 placenta, E12.5 fetal liver and E16.5 fetal liver samples, and lineage-positive cells were depleted using a magnetic system. The Lin^− cells or Lin^− cells cultured with or without FetuA (100 μg ml^−1; 50093-M08H, SinoBiological) or ML216 (25 mM; S0469, Selleck) for 2 h were collected. The cells were then suspended in 1 ml of StemSpan medium (09650, Stem Cell). Hochest33342 (10 µg ml^−1; b2261-25mg, Sigma) and verapamil (50 µM; M14204, AbMole) were added to the cell suspension. The mixture was thoroughly mixed and incubated at 37 °C for 60 min in the dark. Subsequently, 5 µl of 100 µg ml^−1 pyronin Y (213519-1g, Sigma) was directly added to the cells, followed by continuous incubation at 37 °C for another 15 min in the dark. After centrifugation at 300g for 5 min at 4 °C, the cells were suspended in MACS buffer and incubated with biotin-labelled lineage markers (88-7774-75, Thermo Fisher), FITC-labelled Sca-1 (E13-161.7; 122506, BioLegend) and APC-labelled Kit antibodies (2B8; 17-1171-82, Thermo Fisher). Following a 15-min incubation at 4 °C, the cells were washed with PBS, suspended in MACS buffer and incubated with streptavidin–APC–Cy7 (405208, BioLegend). After another 15 min at 4 °C, the cells were washed and resuspended in MACS buffer. The samples were analysed using a Beckman cytoFLEX LX, and the data were analysed using FlowJo (v10).

DNA synthesis analysis of mouse HSPCs by EdU

Pregnant mice were administered intraperitoneal injections of 100 mg kg^−1 EdU (CX000, CellularLab) 2 h before being killed. Cells were collected from E12.5 placenta, E12.5 fetal liver and E16.5 fetal liver and subjected to lineage-positive cell depletion using a magnetic system. The cells were then incubated with biotin-labelled lineage markers (88-7774-75, Thermo Fisher), phycoerythrin–Cy7-labelled Sca-1 (D7; 25-5981-82, Thermo Fisher) and APC-labelled Kit (2B8; 17-1171-82, Thermo Fisher) antibodies. After 15 min at 4 °C, the cells were washed with PBS, suspended in MACS buffer and incubated with streptavidin–phycoerythrin. EdU was detected using the EdU Cell Proliferation Kit with Alexa Fluor 488 (CX002, CellularLab) following the manufacturer’s instructions. The samples were analysed on a Beckman cytoFLEX LX, and the data were analysed using FlowJo (v10).

Cell cycle analysis of mouse HSCs and MPPs by Ki67

Harvested cells from E12.5 placenta, E12.5 fetal liver and E16.5 fetal liver were subjected to lineage-positive cell depletion using a magnetic system. The cells were incubated with biotin-labelled lineage markers (88-7774-75, Thermo Fisher), phycoerythrin–Cy7-labelled Sca-1 (D7; 25-5981-82, Thermo Fisher), APC-labelled Kit (2B8; 17-1171-82, Thermo Fisher), phycoerythrin-labelled CD150 (mShad150; 12-1502-82, Thermo Fisher) and APC–Cy7-labelled CD48 (HM48-1; 103431, BioLegend) antibodies. Following a 15-min incubation at 4 °C, the cells were washed with PBS, suspended in MACS buffer and then incubated with streptavidin–Percpcy5.5. After another 15 min at 4 °C, the cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set Kit (00-5523-00, Invitrogen) according to the manufacturer’s instructions. Subsequently, the samples were incubated with FITC-labelled Ki67 (SolA15; 11-5698-80, Thermo Fisher). After a 30-min incubation at 4 °C, the cells were washed with permeabilization buffer and analysed using a Beckman CytoFLEX LX, and the data were analysed using FlowJo (v10).

RNA synthesis analysis of mouse HSPCs by ethyluridine

Pregnant mice were injected intraperitoneally with 50 mg kg^−1 ethyluridine (2469–25 mg, Lumiprobe) 1 h before being killed. Cells were collected from E12.5 placenta, E12.5 fetal liver and E16.5 fetal liver and subjected to lineage-positive cell depletion using a magnetic system. The cells were then incubated with biotin-labelled lineage markers (88-7774-75, Thermo Fisher), FITC-labelled Sca-1 (E13-161.7; 122506, BioLegend) and APC-labelled Kit (2B8; 17-1171-82, Thermo Fisher) antibodies. After 15 min at 4 °C, the cells were washed with PBS, suspended in MACS buffer and incubated with streptavidin–APC–Cy7 (405208, BioLegend). The ethyluridine was stained using the Cell-Light EU Apollo567 RNA Imaging Kit (C10316-1, RIBOBIO) according to the manufacturer’s instructions. The samples were analysed on a Beckman cytoFLEX LX, and the data were analysed using FlowJo (v10).

Statistics

Statistics analyses were performed using R (v.3.6.3) and GraphPad Prism (v.9.5). n Denotes biological replicates. For violin plots in all panels, the median and quartiles are shown. For boxplots, the mean ± s.d. is shown. For boxplots in Fig. 5c and Extended Data Fig. 9e,i, the median and quartiles are shown. For boxplots in Fig. 5d and Extended Data Fig. 3b, the boxes delimit the minima and maxima, and the horizontal line represents the mean. For survival analysis, the long-rank test was used to compare the difference between groups. For correlation analysis, the Pearson test was used. For comparing two groups, the unpaired Student’s t-tests and Wilcoxon tests were used. P < 0.05 was considered to be significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.