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Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia – Nature Communications

Animal husbandry, timed pregnancies, and transplantation studies

All animal experiments were approved by OHSU or CHOP Animal Care and Use Committees, respectively. Murine Fancd2 is highly homologous to human FANCD2 with 44 exons (Gene bank accession #: BC042619) Fancd2 knockout (Fancd2−/−) mice (C57BL/6 strain, CD45.2 isotype) were generated by a gene-trap method in the laboratory of Alan D’Andrea (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). These Fancd2−/− mice had a phenotype similar to that observed in a previous report (Houghtaling et al.55). Upon gross examination Fancd2−/− mice were consistently smaller than Fancd2+/+ than littermates indicating the general developmental defects in Fancd2−/− mice. Fancd2+/+ and Fancd2−/− fetuses were harvested from timed pregnancies (vaginal plug method) generated by crossing heterozygous Fancd2+/− female with Fancd2+/− male mice. C57/BL6 background, CD45.2 isotype Fancd2 KO mice were bred and used for experiments. Fancd2−/− embryos (E14.5) were utilized for fetal liver studies. Transplantation studies were carried out with B6.SJL-Ptprca Pepcb/BoyJ (CD45.1 isotype) recipients. Fetal livers were dissected from pups and separated by mechanical disruption, filtration, and subsequent red blood cell lysis to get mononuclear cells. Bone marrow was harvested from femurs and tibias of 8–12-week-old Fancd2+/+ and Fancd2−/− animals. Tissues were genotyped for zygosity using primers sets and thermocycler reaction settings listed in supplementary Table-2. For studies involving Ifnar1 KO animals (CD45.2, C57BL/6) were purchased from Jackson Laboratory (colony Stock No. 028288) and 7−8-week-old mice were utilized for timed pregnancies to generate Fancd2−/− Ifnar1+/− embryos (E14.5) by crossing Fancd2+/− Ifnar1+/+ mice with Fancd2+/− Ifnar−/− mice. Mice were housed in a specific pathogen-free (SPF)-grade controlled environment. ALPHA-dri was used as the cage bedding material with the room temperature ranging from 23–27 °C, and humidity of 30–45%. All mice were maintained under a 12-h light/dark cycle and had free access to sterile water and food (5015 (Lab Diet, catalog 0001328, with a metabolizable energy value of 3.59 kcal/g) in wired feeders above the floor of the cage. Animals were euthanized by exposing them to CO2 (carbon dioxide) followed by the physical method of cervical dislocation.

There was no a priori blinding of experimental cohorts at the time of analysis. The experiments described otherwise conformed to the ARRIVE guidelines for reporting animal research.

Transplantation

Harvested and RBC-lysed E12.5 FL cells (5 × 105) were injected via the tail vein in CD45.1 recipients that received 750 cGy using an X-ray irradiator (single dose) At 20 weeks from transplantation, animals were sacrificed, tissues analyzed, and secondary transplantation was performed with injection of 1 × 106 whole bone marrow (WBM) BM cells into 750 cGy irradiated CD45.1 secondary recipients. Peripheral blood from both primary and secondary recipients was analyzed for chimerism, using antibodies against Gr-1, Mac-1, B220, CD3e and DAPI, by FACS. For adult BM transplant experiments, recipients received 1.5 × 106 WT and Fancd2−/− cells at a 1:1 ratio, followed by IP injection of TUDCA in one cohort or an equal volume of 1× sterile PBS.

Mouse embryonic fibroblast (MEF) isolation

E14.5 embryos were harvested from timed pregnancy from Fancd2+/− females to yield Fancd2−/− and Fancd2+/+ embryos. Tissues were minced and trypsinized for 10 min at 37 °C for 10 min. This was followed by washing with complete media and passage of the cell suspension through the 70 μm cell strainers to remove the tissue debris. Cells were then seeded into 0.2% gelatin-coated 100 mm cell culture dishes with 10 ml media (DMEM, 10% FBS, 1% PS) and passaged at ratios of 1:3. When confluent, MEFs were either used directly or stored by cryopreservation in DMSO.

Colony formation unit (CFU) assay

Harvested and RBC-lysed whole FL or lineage-depleted adult BM cells were counted using trypan blue stain and mixed with cytokine-supplemented mouse methylcellulose media (R&D Systems, HSC007). Triplicates were cultured in 3.5 cm dishes at 37 °C. After 7 days, colony numbers were scored under an inverted light microscope.

Immunophenotyping

Single cell resuspended FL or BM mononuclear cells underwent red cell lysis treatment before staining with cKIT at 1:100 concentration (BioLegend 105819), SCA1 (eBioscience 25-5981-81), CD135 (BioLegend), CD48 (BioLegend 103422), and CD150 (BioLegend 115925) antibodies, as well as the lineage antibodies: B220 (BD 553090), GR1 (BioLegend 108408), CD3 (BD 555275), CD4 (BD 553653), CD5 (BD 553023), and TER119 (BD 553673) at the manufacturer’s recommended concentrations. Cells were stained for 30 min on ice, protected from light. Blocking and washing buffer contained 2%FBS/ PBS. For viability, dead cell exclusion staining using DAPI (Thermo 62248, 1μg/ml) was included at a concentration of 1 μg/ml. Flow cytometric analysis was performed using FACS Canto2 and LSR2 instruments (BD Biosciences). For intracellular staining, FL or BM mononuclear cells were stained with surface markers and fixed with 2% PFA for 15 minutes, permeabilized with 0.5% saponin and stained with anti-p53, anti-p53S15 and anti-Cdc7. Samples were acquired with a LSR2 (Becton-Dickinson) and data were analyzed using FlowJo (10.6.1) software to quantify mean fluorescent intensity (MFI). Reagent Supplementaary Table 1 for details.

Immunofluorescence Microscopy

FACS-sorted cells (5–500 × 103) were spun onto glass slides using a cytocentrifuge, followed by incubation with or without Cytoskeletal (CSK) buffer for 10 min at room temperature and fixed in 4% PFA. Permeabilization was performed by the addition of 0.5% Triton and blocking with 3% BSA/PBS at 37 °C for 30 minutes. Primary antibody staining was performed on parafilm at 37 °C or 30 min, and secondary antibodies were used with 1:1000 dilution at 37 °C for 30 min. For nuclear staining, DAPI was used at room temperature for 10 min. For coverslip mounting we used Fluoromount-G (Southern Biotech, 0100). Images were captured on a Core DV microscope (Olympus) and via LSR700 confocal microscope (Carl Zeiss). Images were processed and analyzed with Imaris software (Bitplane). See Supplementary Table 1 for reagent details. To detect ssDNA, we used FlowSight (Amnis) and analyzed with IDEAs software (Amnis).

Quantitative RT-PCR analysis

RNA was isolated using the RNeasy mini and micro kits (QIAGEN). SuperScript IV VILO Master Mix (Invitrogen) was used for cDNA synthesis. For RT-PCR, PowerUp™ SYBR™ Green Master Mix was used from Applied Biosystems. Multiplex real-time PCR was performed with the MYC targets PCR array (QIAGEN) following the manufacturer’s instructions using the Applied Biosystems ViiA™ 7 Real-Time PCR instrument.

EdU/ BrdU cell cycle assay

We used a previously reported assay22 with sequential reagent injection via the tail vein in E13.5 pregnant females with 1 mg of EdU, followed 2 h later by 2 mg of BrdU. After 30 min FLs were harvested and individually processed. Isolated FL mononuclear cells were stained with surface markers (CD150, CD48, c-Kit, Sca-1, Lin) and fixed in 2% paraformaldehyde (PFA) for 15 min, followed by permeabilization with 0.5% saponin and stained with anti-EdU-AF488. To stain with an anti-BrdU antibody, we treated with 20ug of DNAse in PBS (containing Ca2+, Mg2+) at 37 °C for 40 min before staining with BrdU-AF647 (Thermo, B35140). Analysis was performed with FACS on an LSR2 instrument (Becton-Dickinson).

O-Propargyl-puromycin (OPP) assay

For in vivo analysis of rate of translation O-Propargyl-Puromycin (OP-Puro) (Tocris Bioscience; 1416561-90-4) 50 mg/kg body mass; pH 6.4–6.6 resuspended in PBS was injected intraperitoneally into pregnant females at E-14.5 days, 1-hour later mice were euthanized. Dam BM and fetal liver cells were collected as described. RBC lysis was performed with 1× RBC lysis buffer, followed by cell were wash with 1× Ca2+ and Mg2+ (PBS). Stained with combinations of surface lineage markers (CD3, CD4, CD5, B220, Gr-1, Ter119), as well as CD150, CD48, c-Kit, Sca-1, and CD135 (for detail see Table S1) for 30 min on ice, in the dark, followed by washes with 1× PBS and fixation with BD fixative buffer for 15 min in the dark on ice. After washing with 1× PBS, cells were permeabilized in 200 μl PBS supplemented with 3% fetal bovine serum (Sigma) and 0.1% saponin (Sigma) for 5 min at room temperature, protected from light, and then an azide-alkyne click chemistry reaction was performed at room temperature using the Click-iT cell Reaction Buffer Kit. Azide was conjugated to Alexa Fluor 555 (AF555) (Invitrogen, C10642) at 5 μM final concentration for 30 min. Dam bone marrow cells served as unstained control, i.e., they were stained as described above; however, click-iT chemistry reaction was performed without AF555. Samples were acquired by LSR Fortessa and analyzed by FlowJo (10.6.1) software. Results were plotted as OPP median fluorescence intensity (OPP-MFI) using the GraphPad Prism 7.0 software.

MYC protein expression studies

To analyze the levels of MYC expression in Fancd2−/− fetal livers, cells were harvested from E14.5 embryos, and the dam femur BM cells as control. RBC lysis was performed, and cells were stained for 30 minutes on ice with antibodies against cell surface markers (CD3, CD4, CD5, B220, Gr-1, Ter119, CD150, CD48, c-Kit, Sca-1, and CD135; details in Table S1). After staining, cells were washed twice in Ca2+ and Mg2+ free phosphate buffered saline (PBS), fixed, permeabilized, and blocked. Cells were then stained with a primary anti-MYC antibody (Cell Signaling Technologies, D84C12) on ice for 30 minutes, followed by PBS washing and staining with a secondary antibody (Invitrogen,12-4739-81) for 30 min on ice. Cells were washed twice with PBS, and samples were acquired on LSR-Fortessa instrument. Data was analyzed by FlowJo (10.6.1) software and results were plotted as median fluorescent intensity using GraphPad Prism 7.0 software.

Tetraphenylethene maleimide (TPE-MI) assay

To determine unfolded protein levels, fetal liver cells and bone marrow cells were harvested from the E-14.5 embryos and tibia and femur of the dams, respectively, followed by staining with cell surface markers described above. After surface epitope staining, cells were washed twice in Ca2+ and Mg2+ free phosphate buffered saline (PBS), followed by Tetraphenylethene maleimide (TPE-MI; stock 2 mM in DMSO) diluted in PBS (50 mM final concentration). This was added to each sample, and samples were incubated at 37 °C for 45 minutes. Samples were washed in PBS and fixed with fixative buffer for 15 min on ice then samples were washed twice with PBS and acquired by flow-cytometry using an LSR-Fortessa instrument. We cultured lineage-depleted bone marrow cells with 1 μM of Thapsigargin (Med Chem Express, HY-13433) for 4 h in vitro as a positive control; unstained cells served as an experimental negative control.

Proteasome activity assay

LSK cells were sorted from the total fetal liver and dams BM cells after RBC lysis and surface staining and sorted cells were plated in triplicates (5 × 103 cells in 100 μl/well) from each sample into the 96 well plates and 100 μl of reconstituted proteasome -Glow chymotrypsin like cell-based assay substrate (Promega) were added to each well and contents were mixed properly by plate shaker for 2 min at 700 rpm. After 10 min of incubation at room temperature luminescence was measured by Tecan infinite-M200 multimode plate reader and results were plotted by GraphPad Prism 7.0 software.

Proteostat assay

Fetal livers from WT and Fancd2−/− FL were isolated and cells were sorted for KSL immunophenotype (Lin, c-kit+, Sca-1+) on a FACS -Aria Fusion sorter. Sorted cells were then seeded onto positively charged glass slides, fixed, permeabilized and incubated with Enzo Proteostat Detection Reagent (Enzo Life Sciences, cat #: ENZ-51035-K100). A DAPI nuclear counterstain was applied. Slides were mounted on coverslips and imaged with a Nikon Eclipse 50i microscope, using Micro-Manager analysis software.

TUDCA (Tauroursodeoxy-cholic acid) treatment

TUDCA (Sigma-Aldrich) was dissolved in sterile PBS and that was intraperitoneally (IP) injected (7.5 mg/kg) into pregnant mice serially from embryonic day 10.5–14.5 of pregnancy and E14.5 embryos were harvested one hour after TUDCA injections for TPE-MI analysis. For ex vivo culture Fancd2−/− fetal liver cells were cultured for 18 h in Stem Span media, supplemented with IL-6 (500 μg), IL-3 (500 μg) and SCF (2500 μg), in 50 ml Stem Span and 1% PS. For adult BM transplant experiments, recipient animals received daily serial TUDCA (IP) injections followed by two injections per week for 10 weeks at 7.5 mg/kg.

Single-cell RNA sequencing

DNA was extracted from a small amount of tissue from each embryo harvested at E13.5 using the KAPA Mouse Genotyping Kit (Roche, KK7352). The genotype and sex of each embryo was then determined via end-point PCR with the Hot Start Taq Master Mix Kit (Qiagen, 203443) or qRT-PCR with Power SYBR™ Green PCR Master Mix (Applied Bioscience, 4367659), respectively (Supplementary Table 4). Fancd2−/− embryos were identified, along with sex-matched WT littermate controls where possible. Fetal livers were dissected and mechanically digested in 900 µL ice-cold PBS + 0.04% BSA (PBS+) via pipetting, before shaking at 220 rpm for 10 min at 37 °C in 0.3% collagenase I (StemCell Technologies, 07416). Samples were washed with 5 mL ice-cold PBS+ and centrifuged, followed by resuspension in 2 mL ice-cold freshly prepared 1× RBC lysis buffer (Invitrogen, 00-4300-54). They were incubated on ice for 1 min and pelleted again, followed by resuspension in 1 mL ice-cold PBS+, and left on ice until staining for FACS. All centrifugation was performed at 400 × g for 5 min at 4 °C, and all cell pellet resuspension was performed using wide-bore pipette tips. To safeguard adequate representation of smaller cell populations in the dataset, we first selectively depleted FL cells of the predominant Ter119 expressing erythroid cells before submission for library construction and sequencing. Samples were centrifuged and resuspended in a 1:100 dilution of FITC anti-mouse TER-119 antibody (BioLegend, 116206) with PBS and incubated on ice for 20 minutes, protected from light. After washing with 1 mL ice-cold PBS+, samples were centrifuged and resuspended in 300 µL ice-cold PBS+ with 5 µL propidium iodide (PI) solution (Miltenyi Biotec, 130-093-233). After at least 5 minutes on ice, protected from light, samples were sorted for live Ter119-negative cells using a FACS-Jazz instrument, and a cell count with Trypan blue was performed to assess cell viability. Samples were suspended in PBS at a concentration of 1000 cells/µL and submitted to the Children’s Hospital of Philadelphia Center for Applied Genomics for same-day library generation.

Library generation and 10X genomics sequencing

For library preparation, standard procedure was followed as written in the Chromium Single Cell 3’ Reagent Kits User Guide (v3.1 Chemistry). Library QC was performed using the Agilent Tape Station (High Sensitivity D1000 Screen Tape, 5067-5584; Reagents, 5067-5585) and KAPA Library Quantification Kit (Roche, 07960336001), followed by sequencing on the Illumina NovaSeq 6000 using a S2 100 cycles flow cell v1.5.

Sequencing data analysis

Raw data was processed with Cell Ranger to obtain a gene-cell read count matrix. Data were filtered using Seurat version 4.0.1 to only include: genes detected in at least 3 cells; cells with 200–6000 detected genes cells with reads from mitochondrial genes less than 5% of total reads. Cells from all libraries were pooled together using the Seurat standard integration procedure, and the top 2000 genes with the highest variance. Cells were clustered by applying the PCA method to normalized read counts. Cell clusters were visualized by UMAP and manually assigned to known cell types based on their expression level of marker genes. Read counts of cells from the same biological replicate and cell type were pooled together to generate a gene-sample read count matrix. DESeq2 was applied to all gene-sample matrixes to analyze the differential gene expression between two genotypes. The adjusted fold changes calculated by DESeq2 were used to rank genes for gene set enrichment analysis.

Resource availability

Further information/requests about resources involved in this study should be directed to and will be fulfilled by the Lead Contact, Peter Kurre (kurrep@chop.edu).

Statistical analysis

All numerical results were expressed as mean ± SEM. As appropriate, two-tailed Student’s t tests, Welch test and one-way ANOVA were performed for statistical analyses. All analyses were performed with GraphPad Prism7.0 software. For GSEA analysis both NES and P values are statistical outputs from the GSEA run themselves.

Reporting summary

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