TGFβ pathway inhibitors rescue hematopoiesis in FA models

The canonical TGFβ pathway is upregulated in the bone marrow of patients with FA [7, 10]. Accordingly, TGFβ pathway inhibitors, targeting the TGFβ Receptor I (TGFβ RI), such as Galunisertib and LSN3301240, were initially tested for their ability to rescue hematopoiesis in FA models. As predicted, these inhibitors increased the in vitro colony forming unit (CFU) clonogenic capacity of Lin^- cells from Fancd2^-/- mice (Fig 1A), correlating with a decrease in the phosphorylation of SMAD2/3 in the presence of the TGFβ1 and TGFβ3 in WT and Fancd2^-/- cells (Fig 1B). The inhibitors also increased the CFU numbers in the cells from WT mice, however, the increase in CFU was more significant in cells from Fancd2^-/- mice. Consistent with previous studies with other TGFβ pathway inhibitors [7, 10], Galunisertib and LSN3301240 also improved the clonogenic capacity of FA-like HSPCs. These FA-like cells were generated by infection of CD34⁺ cord blood cells with a lentivirus expressing a shRNA against FANCD2 (S1A Fig). LSN3301240 also partially rescued Fancd2^-/- stromal cell lines from MMC-induced genotoxicity (Fig 1C), although this effect was not observed with Galunisertib (S1B Fig). We next tested LSN3301240 for its ability to protect LT-HSC from DNA damage caused by pI:pC, an agent known to induce physiologic stress [21]. We co-injected WT or Fancd2^-/- mice with pI:pC and LSN3301240, and the amount of DNA damage in sorted LT-HSC (identified as Lin^-LSK CD150⁺CD48^- cells) was quantified using the comet assay (Fig 1D). Treatment of Fancd2^-/- mice with LSN3301240 significantly reduced the amount of DNA damage induced by pI:pC in LT-HSCs (Fig 1E). Taken together, inhibition of the TGFβ pathway rescued several of the hematopoietic features of FA bone marrow cells.

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Fig 1. TGFβ pathway inhibitors rescue hematopoiesis in FA models.

(A) TGFβ pathway inhibitors Galunisertib and LSN3301240 increase the clonogenic capacity of Lin^- cells from bone marrow of Fancd2^-/- mice measured in a CFU assay. Lineage negative primary bone marrow cells from wild-type (WT) or Fancd2^-/- mice were cultured in methylcellulose medium containing increasing doses of Galunisertib or LSN3301240 for 7 days and hematopoietic colonies (CFUs) were counted for evaluating clonogenic growth of progenitors. (B) Western blots of the lysates from Lin⁻ cells from bone marrow of wild-type and Fancd2^-/- mice cultured for 2 hours in the presence of TGFβ1 (5 ng/mL) or TGFβ3 (5 ng/mL) with or without LSN3301240. Levels of phospho-Smad2 (measured by two different antibodies, one against phospho-Smad2 and the other against both phospho-Smad2 and Smad3) are shown. Quantification relative to basal conditions and the loading control is shown below every lane. Pink indicates samples treated with LSN3301240. (C) Stromal cell lines generated from WT and Fancd2^-/- mice were cultured in the presence of LSN3301240 and Mitomycin C (MMC) and survival was determined. LSN3301240 partially rescued the MMC sensitivity characteristic of FA cell lines. (D) Schematics showing treatment of WT and Fancd2^-/- mice in vivo with pI:pC + LSN3301240. Wild-type (WT) or Fancd2^-/- mice (KO) were injected with pI:pC along with LSN3301240 and 48 hrs after the exposure, DNA damage was analyzed in bone marrow LT-HSCs using a comet assay. (E) Comet assay on sorted LT-HSCs showing that pI:pC increases the DNA damage, measured by tail length, of LT-HSCs, whereas co-treatment with LSN3301240 reduces DNA damage in LT-HSCs from Fancd2^-/- mice. Data in (A) and (C) are represented as mean ± SEM. Data in (E) are represented as boxplots. p values of 0.01 to 0.05 were considered significant (*), p values of 0.001 to 0.01 were considered very significant (**) and p values of < 0.001 were considered extremely significant (***, ****). See also S1 Fig.

https://doi.org/10.1371/journal.pgen.1010459.g001

Double knockout of the canonical TGFβ pathway and Fancd2 results in embryonic lethality

Hyperactive TGFβ pathway signaling causes overexpression of genes in the low-fidelity NHEJ pathway of DNA repair, thereby contributing to the bone marrow dysfunction observed in FA mice and FA patients. Since pharmacological inhibition of the TGFβ pathway improves bone marrow dysfunction, we hypothesized that a mouse model with disruption of Fancd2 and Smad3, the canonical mediator of the TGFβ pathway, would overcome this FA hematopoietic defect and perhaps increase the survival of DKO newborn mice. To test this hypothesis, we generated four different double knockout (DKO) crosses of Smad3^+/- and Fancd2^+/- mouse strains. Two different Smad3^+/- and Fancd2^+/- mice strains on two different genetic backgrounds (129/Sv and C57BL/6) were bred to obtain 4 strains of Smad3^-/- Fancd2^-/- DKO mice, named Smad3 Fancd2-129B6F2;

Smad3 Fancd2-129129F2; Smad3 Fancd2-B6B6F2 and Smad3 Fancd2-B6129F2 (Figs 2A and S2A). Phenotype for these crosses can be seen in S1 Table.

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Fig 2. Smad3 loss is synthetic lethal with Fancd2 loss and surviving DKO mice do not have improved adult hematopoiesis.

(A) Two different Smad3^+/- and Fancd2^+/- mouse strains on two different genetic backgrounds (129/Sv and C57BL/6) were bred to obtain 4 strains of Smad3^-/- Fancd2^-/- double Knockout (DKO) mice: Smad3 Fancd2-129B6F2; Smad3 Fancd2-129129F2; Smad3 Fancd2-B6B6F2 and Smad3 Fancd2-B6129F2 (See also S2A Fig). (B) Breeding frequency of DKO mice was significantly reduced in all 4 background combinations compared to the expected 6.5% frequency of DKO births from breeding double heterozygotes, suggesting embryonic lethality. For Smad3 Fancd2-129B6F2; Smad3 Fancd2-129129F2; Smad3 Fancd2-B6B6F2 and Smad3 Fancd2-B6129F2, we tested whether the frequency of births is significantly different from an expected frequency of 1 in 16, using the two-sided proportional test. The p-values are adjusted for multiple tests with the Bonferroni method. The adjusted p-values for the four breeds are <0.0001, 0.0028, <0.0001 and 0.0044 respectively, so the birth frequencies are all significantly smaller than 1/16 (i.e. 0.0625). The exact 95% confidence interval for the four breeds are respectively (0.0251, 0.0462), (0.0187, 0.0468), (0.0006, 0.0192) and (0.0011, 0.0325). (C) Reduced frequency of bone marrow hematopoietic stem cell (HSC) numbers in Smad3 Fancd2-129B6F2 mice compared to parental strains. (D) In vitro LTBMC assay showing that bone marrow from adult Smad3 Fancd2-129B6F2 (Fancd2^-/-Smad3^-/-) mice have a reduced production of hematopoietic progenitors, similar to a Fancd2^-/- (Fancd2^-/- Smad3^+/+) mouse bone marrow genotype. Results are presented as cumulative day 14 CFU-GEMM forming cells. (E) Competitive repopulation capacity of the bone marrow in transplant assays showing that hematopoietic cells derived from adult Smad3 Fancd2-129B6F2 (Fancd2^-/-Smad3^-/-) mice have a defective hematopoiesis, similar to Fancd2^-/- (Fancd2^-/- Smad3^+/+) mice. (F) Survival of stromal cell lines derived from the bone marrow of adult mice showing that cells from Smad3 Fancd2-129B6F2 (Fancd2^-/-Smad3^-/-) mice are hypersensitive to MMC. (G) Survival of stromal cell lines derived from bone marrow of adult mice showing that cells from Smad3 Fancd2-129B6F2 (Fancd2^-/-Smad3^-/-) mice are hypersensitive to irradiation. (H) Western blots of the lysates from stromal cell lines derived from bone marrow of adult mice showing that cells from Smad3 Fancd2-129B6F2 (Fancd2^-/-Smad3^-/-) mice overexpress the NHEJ proteins DNA-PKcs and 53BP1, and RAD51 a mediator for homologous recombination. A representative blot from three independent experiments is shown. Quantification relative to wild type in basal conditions and the loading control is shown below every lane. Pink indicates irradiated samples (10 Gy). Cell lysis for western blot was performed 4 h after IR. Data in (B) and (C) are represented as bar plots. Data in (D), (E), (F) and (G) are represented as mean ± SEM. p values of 0.01 to 0.05 were considered significant (*), p values of 0.001 to 0.01 were considered very significant (**) and p values of < 0.001 were considered extremely significant (***, ****). See also S2 Fig.

https://doi.org/10.1371/journal.pgen.1010459.g002

Surprisingly, for every tested DKO breed we observed a decrease in the number of weaned DKO pups (Fig 2B), demonstrating that embryonic lethality was characteristic of DKO embryos, regardless of the mouse strain. These findings suggested that overexpression of the TGFβ pathway is paradoxically required for the survival of embryos with a FA pathway deficiency. Since the Smad3 Fancd2-129B6F2 cross yielded the highest number of surviving DKO mice we analyzed the hematopoietic function of Smad3 Fancd2-129B6F2 adult mice.

Surviving DKO mice exhibit dysfunctional adult hematopoiesis

Smad3 Fancd2-129B6F2 adult mice were next evaluated for their hematopoietic function, compared to WT, Fancd2^-/-, and Smad3^-/- adult mice. The Smad3 Fancd2-129B6F2 adult mice and the Fancd2^-/- mice exhibited a reduced number of hematopoietic stem cells, compared to the WT and Smad3^-/- mice (Fig 2C). Similarly, hematopoietic stem cells from adult Smad3 Fancd2-129B6F2 mice or Fancd2^-/- mice were impaired in their production of hematopoietic progenitors, based on LTBMC (Stroma-dependent long-term bone marrow culture) assays (Fig 2D), compared with the WT and Smad3^-/- mice. Similar results were observed in LTBMC assays for the Smad3 Fancd2-129129F2; Smad3 Fancd2-B6B6F2 and Smad3 Fancd2-B6129F2 crosses (S2B Fig). In vivo repopulation assays also revealed a reduced repopulation capacity of Smad3 Fancd2-129B6F2 and Fancd2^-/- hematopoietic stem cells (Fig 2E). A similar reduction in repopulation capacity was observed for hematopoietic cells derived from Smad3 Fancd2-129129F2 mice (S2C Fig).

In vitro assessment of DNA repair capacity in stromal fibroblast lines demonstrated that Smad3 Fancd2-129B6F2 cells are hypersensitive to MMC, similar to the phenotype of Fancd2 deficient cells (Fig 2F), with modest differences likely due to their genetic backgrounds, Smad3 Fancd2-129129F2; Smad3 Fancd2-B6B6F2 and Smad3 Fancd2-B6129F2 derived stroma were also sensitive to MMC (S2D Fig). These results were also surprising since Fancd2-deficient cells exposed to the TGFβ pathway inhibitors (Galunisertib or LSN3301240), had relative MMC resistance (Fig 1C). Smad3 Fancd2-129B6F2 cells were also hypersensitive to irradiation (Fig 2G), implying that Smad3 Fancd2-129B6F2 cells have multiple DNA repair defects.

We hypothesized that the sensitivity to irradiation in the Smad3 Fancd2-129B6F2 cells is due to deficiency in the expression of NHEJ proteins; however, after irradiating the cells we observed that Smad3 Fancd2-129B6F2 cells express DNA-PKcs and 53BP1, the prototypical proteins mediating NHEJ (Fig 2H). Smad3 Fancd2-129B6F2 cells not only had upregulation of NHEJ proteins but also expressed high levels of RAD51, a protein known to mediate HR (Fig 2H). Nonetheless, the DKO cells were unable to tolerate IR.

Upregulation of TGFβ pathway genes and error-prone DNA repair pathway genes in FA mouse embryos

We hypothesized that the few surviving DKO pups were the exception to the generalized lethality observed for the DKO crosses. We reasoned that the canonical TGFβ pathway might have a critical role in allowing Fancd2^-/- embryos to survive during mid-gestation. To test this hypothesis, pregnant females were sacrificed at day E12.5, uterine horns were dissected, and the recovered embryos were genotyped (Fig 3A). The frequencies of the different genotypes in the Smad3 Fancd2-129B6F2 breeding at day E12.5 recapitulated the predicted normal Mendelian frequency (Figs 3B and S3A). The expected Mendelian ratio for the Smad3^-/-Fancd2^-/- DKO mice was preserved at this early embryonic stage of development, especially for the Smad3 Fancd2-129B6F2 breeding (Figs 3C and S3B). However, no DKO embryos were observed at day E14.5 (S3C Fig), further demonstrating that Smad3 and the canonical TGFβ pathway are essential for the survival of FA embryos from day E12.5 onwards to birth. Of note, at day E14.5 of gestation, Fancd2^-/- embryos already displayed defective hematopoiesis, as determined by reduced hematopoietic colony numbers in vitro, based on a CFU assay of cells derived from mouse fetal liver (S3D Fig).

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Fig 3. Fancd2^-/- embryos overexpress the TGFβ pathway and error-prone DNA repair genes.

(A) Fancd2/Smad3 heterozygous littermates were crossed to breed. Pregnant females were sacrificed at day E12.5 of gestation, uterine horns were dissected, and embryos genotyped. RNA from embryos was analyzed for expression of genes in the TGFβ pathway and DNA repair pathways. (B) Smad3 Fancd2-129B6F2 breeding has a Mendelian distribution at day E12.5, showing that Smad3 Fancd2-129B6F2 embryos can survive until day E12.5 of embryonic development (n = 141 embryos). (C) The frequency of Smad3 Fancd2-129B6F2 embryos at day E12.5 yields the expected Mendelian ratio, however the frequency of weaned Smad3 Fancd2-129B6F2 pups is halved, suggesting later lethality during embryonic development. (D) Gene expression analysis of E12.5 embryos. Left. Compared to WT embryos, Fancd2^-/- embryos overexpress TGFβ pathway-related genes and overexpress the ligands for activation of the TGFβ pathway, Tgfb1 and Tgfb3, as well as their receptors Tgfbr2 and Tgfbr3. The oncogene Myc was also found to be overexpressed in Fancd2^-/- embryos. Right. Compared to WT embryos, Fancd2^-/- embryos overexpress genes related to alternative DNA repair pathways, including Prkdc, Mad2l2 and Parp1, as well as negative regulators of cell cycle progression, including Trp53 (p53) and Cdkn1a (p21). Red dots indicate differentially upregulated genes with respect to wild type embryos. Blue dots indicate differentially downregulated genes with respect to wild type embryos. Data in (B) and (C) are represented as bar plots. Data in (D) are represented as volcano plots. p values of 0.01 to 0.05 were considered significant (*), p values of 0.001 to 0.01 were considered very significant (**) and p values of < 0.001 were considered extremely significant (***, ****). See also S3 Fig.

https://doi.org/10.1371/journal.pgen.1010459.g003

In order to explore the relevance of the canonical TGFβ-Smad3 pathway during FA ontogenesis, we used targeted RNA sequencing and transcriptional profiling of the TGFβ pathway in Fancd2^-/- embryos (Fig 3D, left). Whole embryos were collected at day E12.5, and the isolated total body RNA was evaluated. As predicted, the mRNAs encoding several components of the TGFβ superfamily were upregulated in Fancd2^-/- embryos, compared to WT embryo controls. These upregulated mRNA transcripts included the Tgfβ1 and Tgfβ3 mRNAs, encoding ligands at the apex of the TGFβ pathway. The mRNAs encoding the receptors for these ligands, Tgfbr2 and Tgfbr3, were also upregulated in the Fancd2-deficient embryos. This pattern of transcriptional upregulation further suggests a requirement of TGFβ pathway signaling during the embryogenesis of the Fancd2^-/- mice. Of note, mRNAs with opposing functions were also upregulated (Fig 3D), including Smad7, known as an Inhibitory-Smads, and Myc, a proto-oncogene that stimulates cell proliferation and overcomes TGFβ growth suppression [6]. Taken together, these data suggest that compensatory negative feedback mechanisms, counteracting the hyperactive TGFβ pathway, are also present at the organismal level during development of embryos with an FA pathway deficiency.

Overexpression of NHEJ genes, has also been observed in FA cells, and several reports have demonstrated that the canonical TGFβ pathway upregulates these pathways [7, 10, 22]. Since FA is a DNA repair deficiency syndrome, we used targeted RNA sequencing to evaluate the expression of DNA repair genes at day E12.5 of embryonic development (Fig 3D, right). Interestingly, Trp53 and Cdkn1a, which encodes p21, are overexpressed in the whole embryo, confirming that DNA stress responses leading to cell cycle arrest and apoptosis are highly active during development of an FA embryo. Previous studies had indicated that expression of these genes is upregulated in the hematopoietic compartment of human FA embryos [5]. Among the upregulated error-prone DNA repair genes in FA embryos were Prkdc, Mad2l2, Trp53bp1, Parp1 and Lig3 (Fig 3D). Importantly, overexpression of low-fidelity NHEJ and alternative error-prone genes has detrimental consequences for the FA adult hematopoiesis. However, the access to high-capacity but low-fidelity DNA repair pathways during rapid cell proliferation stages, such as embryogenesis, appears to be critical for ensuring the survival of an embryo with a deficiency in the FA pathway. We speculate that, although toxic for adult hematopoiesis, the activation of these alternative DNA repair pathways must be essential for viable gestation of FA embryos. The expression of TGFβ genes and DNA repair genes was also assessed in Smad3^-/- and Smad3 Fancd2-129B6F2 embryos at day E12.5 (S3E Fig). In comparison to wild type, the Smad3^-/- embryos exhibited increased expression of the TGFβ pathway genes Tgfb1, Myc, and Bmp3 and increased expression of the DNA repair genes Mad2l2 and Trip13. In comparison to wild type the Smad3 Fancd2-129B6F2 embryos continue overexpressing member of the TGFβ pathway, including the Tgfb1 ligand. Finally, in comparison to the Fancd2^-/-, Smad3 Fancd2-129B6F2 embryos display downregulation of multiple DNA repair genes.

Activation of the non-canonical ERK pathway in a subset of Smad3 Fancd2-129B6F2 embryos

Extensive literature has shown that cells that lose the canonical TGFβ signaling pathway can retain responsiveness to TGFβ ligands and in some cases become dependent of non-canonical TGFβ pathways (Finnson et al., 2020). We found in our gene expression analysis, that Smad3 Fancd2-129B6F2 embryos overexpress the Tgfb1 ligand (S3E Fig middle panel), and found also that Smad3 Fancd2-129B6F2 adults have increased levels of Tgfb ligands in peripheral blood plasma (S4A Fig). These results suggest that the TGFβ ligands are not lost in our DKO mice and could signal intracellularly through non-canonical TGFβ pathways, and contribute to retention of the FA phenotype. We therefore hypothesized that the Smad3 Fancd2-129B6F2 embryos surviving to the adult stage might activate intracellular mediators of the non-canonical TGFβ pathway, such as the ERK1 pathway. This non-canonical pathway might in turn stimulate the expression of essential genes in the NHEJ pathway in cells deficient in Smad3. This survival pathway would maintain a hyperactive TGFβ-NHEJ axis in the surviving Smad3 Fancd2-129B6F2 mice and could account for their subsequent defect in hematopoietic improvement.

Irradiation is known to activate the TGFβ pathway [16]. We therefore irradiated stromal cells from the different DKO backgrounds and measured phosphorylation of Smad3 (p-Smad3). WT and Fancd2^-/- cell lines upregulated p-Smad3, while the Smad3^-/- and Smad3 Fancd2-129B6F2 cell lines did not. However, phosphorylation of ERK (p-ERK), an alternative signaling mediator of the TGFβ pathway, was observed in Smad3^-/- and Smad3 Fancd2-129B6F2 mice, suggesting that activation of the non-canonical TGFβ-ERK pathway occurs in the surviving Smad3 Fancd2-129B6F2 mice and might contribute to their persistent bone marrow dysfunction (Figs 4A and S4B). Inactivation of the SMAD3 pathway was further confirmed by testing SNAIL, a SMAD3 target, that as expected, was reduced in the Smad3^-/- mutant cell line [23]. Interestingly, in the DKO cell line, SNAIL is present, we speculate this might be due to regulation of SNAIL by other transcription factor in the surviving DKO context (S4C Fig). A similar response to irradiation was observed in the Smad3 Fancd2-129129F2 stroma (S4D Fig), the Smad3 Fancd2-B6B6F2 stroma (S4E Fig) and the Smad3 Fancd2-B6129F2 stroma (S4F Fig).

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Fig 4. Smad3 Fancd2-129B6F2 embryos with ERK pathway activity express NHEJ components.

(A) Western blot of the lysates from adult bone marrow stroma cells of mice showing that irradiation activates the canonical phosphorylation of Smad3 in bone marrow stroma of WT and Fancd2^-/- mice and activates the non-canonical ERK1/2 phosphorylation in Smad3^-/- and DKO mice stroma. Quantifications relative to wild type in basal conditions and the loading control are shown below every lane. Pink indicates the irradiated cultures per cell line. (B) Western blot of the lysates from E12.5 fetal liver showing that a subset of Smad3 Fancd2-129B6F2 embryos have higher levels of the phospho-ERK protein, suggesting activation of the non-canonical TGFβ pathway. High phospho-ERK embryos have a reduced expression of CD45 and CD41 (CD45 and CD41 were used as surrogates for indication of active hematopoiesis in the fetal liver). High phospho-ERK embryos have higher expression of NHEJ related proteins, DNA-PK and 53BP1. Two western blots are depicted showing a total of two wild type fetal livers, two Fancd2^-/- fetal livers, two Smad3^-/- fetal livers, and six Smad3 Fancd2-129B6F2 fetal livers. Quantifications relative to wild type in basal conditions and the loading control per gel are shown below every lane. Red indicates Fancd2^-/- fetal livers, pink indicates Smad3 Fancd2-129B6F2 fetal livers with high phospho-ERK levels. (C) Left. Representative scatter plots of variant allele fractions (VAFs) of the 129/Sv germline variants across the genomes of the analyzed embryos (n = 2 wild type, n = 3 Fancd2^-/-, n = 2 Smad3^-/-, n = 6 Smad3 Fancd2-129B6F2). Long chromosomal segments with known strain origins are clearly observed. Homozygous reference (C57BL/6) segments are those with VAFs around zero (red); homozygous 129/Sv segments are those with VAFs around one (blue); and heterozygous segments of both strains are those with VAFs between zero and one (green). Wild type and Smad3^-/- embryos show a balanced heterozygosity with VAFs of heterozygous sites clustering around 0.5. Fancd2^-/- and Smad3 Fancd2-129B6F2 embryos show pronounced genomic instability with VAFs of heterozygous sites deviating markedly from 0.5. Right. Green inset summarizes the density of VAFs in the heterozygous 129/Sv germline. (D) Heatmap for gene expression showing that two types of E12.5 Smad3 Fancd2-129B6F2 embryos exist. Half of the studied E12.5 Smad3 Fancd2-129B6F2 embryos have a Bax^High gene expression profile (suggesting lethality) and the other half have a Bax^Low gene expression profile (suggesting survivorship). When profiled for DNA repair related genes, these two groups of E12.5 Smad3 Fancd2-129B6F2 embryos seem to have mutually exclusive gene expression profiles, with down-regulation of alternative error-prone and low fidelity DNA repair genes in the Bax^High embryos, an effect similar to what has been previously ascribed to pharmaceutical inhibition of the TGFβ pathway. Black arrowhead indicates Prkdc, the gene codifying for DNA-PKcs. Data in (C) are represented as variant allele frequency (VAF). Data in (D) are represented as heatmap. See also S4 Fig.

https://doi.org/10.1371/journal.pgen.1010459.g004

Western blot analysis using protein lysates from E12.5 fetal livers confirmed that a subset of Smad3 Fancd2-129B6F2 embryos have high levels of phospho-ERK1/2 (Fig 4B). As a surrogate for hematopoietic activity in the fetal liver, we used the CD45 and CD41 markers and found that fetal livers with high phospho-ERK1/2 levels have reduced fetal hematopoiesis and have increased levels of DNA-PKcs and 53BP1, classic NHEJ proteins (Fig 4B).

DNA damage has been shown to leave a scar in the genome of DNA repair deficient cell lines [24], we therefore hypothesized that Fancd2^-/- embryos would have an enhanced genomic instability and tested the same condition in the Smad3 Fancd2-129B6F2 embryos. DNA was extracted from E12.5 embryos and subjected to whole exome sequencing followed by detection of variants. Germline variants were identified, and scatter plots of variant allele fractions (VAFs) (Fig 4C), as well as plots of copy number variants (CNVs) (S4G Fig) were constructed. The wild type and Smad3^-/- embryos show a balanced heterozygosity with VAFs of heterozygous sites clustering around 0.5, whereas the Fancd2^-/- and Smad3 Fancd2-129B6F2 embryos show pronounced genomic instability with VAFs of heterozygous sites deviating markedly from 0.5 (S4C). The CNV analysis showed DNA segments with normal copy ratios close to 1 in wild type embryos, whereas the other genotypes display DNA segments with copy ratios markedly different from 1, especially Smad3 Fancd2-129B6F2, indicating high degree of aneuploidy (S4G Fig). This data shows that the deficiency of FA pathway incites genomic instability, which starts early in utero in FA embryos and depletion of Smad3 failed to correct this imbalance.

Previous studies have shown that the TGFβ pathway positively regulates the expression of NHEJ genes [10, 16]. We therefore hypothesized that loss of NHEJ activity would occur in embryos with Smad3 knockout. Interestingly, when we explored in depth the gene expression profile of Smad3 Fancd2-129B6F2 embryos at day E12.5, we identified two types of Smad3 Fancd2-129B6F2 embryos with opposing gene expression patterns (Fig 4D). Approximately half of the embryos overexpressed the pro-apoptotic Bax gene (Bax^High Smad3 Fancd2-129B6F2), indicating a survival disadvantage. The other half of embryos downregulated Bax (Bax^Low Smad3 Fancd2-129B6F2) and had a presumed survival improvement, based on the number of surviving Smad3 Fancd2-129B6F2 newborns (See Figs 2B and 3B). Of note, the Bax^High Smad3 Fancd2-129B6F2 embryos downregulated the low-fidelity DNA repair pathway genes, such as Prkdc that codes for DNA-PKcs, which were otherwise upregulated in Fancd2^-/- embryos (Fig 3D). This result was expected and was consistent with previous studies showing downregulation of NHEJ pathway genes following TGFβ pathway inhibition in FA cells [10]. The Bax^Low Smad3 Fancd2-129B6F2 embryos had the opposite gene expression profile. Interestingly, these Bax^Low Smad3 Fancd2-129B6F2 embryos expressed high levels of NHEJ mRNAs, such as Prkdc, despite the absence of the canonical Smad3-driven TGFβ pathway (Fig 4D).

As we showed that the non-canonical ERK pathway is activated in a subset of Smad3 Fancd2-129B6F2 embryos and in the surviving Smad3 Fancd2-129B6F2 mice, we explored the presence of TGFβ ligands in the supernatant from our LTBMC long-term cultures using an ELISA assay. In this assay we discovered that Smad3 Fancd2-129B6F2 cultures secrete higher levels of TGFβ ligand than the other genotypes (S4D Fig), suggesting that TGFβ ligand production is not lost in the surviving Smad3 Fancd2-129B6F2 mice and is being transduced through the ERK non-canonical pathway.

Taken together, these findings suggest that knockdown of Smad3 in a FA background reduces NHEJ activity and promotes hematopoietic activity; however, the NHEJ pathway is still critical for the survival of FA embryos. Accordingly, the DKO embryos survive by activating an alternative non-canonical TGFβ signaling pathway, through the ERK kinase, in order to upregulate NHEJ.

Inhibition of the non-canonical TGFβ pathway rescues Smad3 Fancd2-129B6F2 cells from genotoxicity

We next reasoned that inhibition of the non-canonical ERK pathway in the Smad3 Fancd2-129B6F2 stromal cells derived from adult Smad3 Fancd2-129B6F2 mice would block NHEJ activity. Indeed, inhibition of the non-canonical ERK pathway with the MEK inhibitor (MEKi) PD0325901 resulted in a reduction of DNA-PKcs and 53BP1 protein levels. Interestingly, DNA-PKcs reduction was exclusively observed in the Smad3 Fancd2-129B6F2 cells but not in the Fancd2^-/- cells, suggesting that DNA-PKcs in Fancd2^-/- cells is not under the control of ERK and the non-canonical pathway (Fig 5A).

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Fig 5. Inhibition of ERK in surviving Smad3 Fancd2-129B6F2 mice stroma reduces NHEJ proteins and improves DNA repair.

(A) Western blots of the lysates from bone marrow stromal cells showing that inhibition of ERK phosphorylation with the MEK inhibitor PD0325901 reduces the levels of DNA-PKcs in DKO cells but not in Fancd2^-/- cells. A representative blot from three independent experiments is shown. Quantification relative to wild type in basal conditions and the loading control is shown below every lane. Pink indicates samples treated with MEKi PD0325901. (B) Quantitation of γH2AX foci by immunofluorescence showing that the MEK inhibitor PD0325901 reduces the number of γH2AX foci in Fancd2^-/- cells and DKO cells. (C) Quantitation of DNA-PKcs foci by immunofluorescence showing that the MEK inhibitor PD0325901 significantly reduces the number of DNA-PKcs foci in DKO cells. (D) Representative images of γH2AX foci and DNA-PKcs foci in Fancd2^-/- and DKO stromal cells after exposure with MEK inhibitor PD0325901. (E) Quantitation of 53BP1 foci by immunofluorescence showing that the MEK inhibitor PD0325901 significantly reduces the number of 53BP1 foci in DKO cells. (F) Survival curves of bone marrow stromal cells showing that the MEK inhibitor PD0325901 rescues Fancd2^-/- and Smad3 Fancd2-129B6F2 stromal cells from MMC toxicity. Response of the WT and Smad3^-/- cell lines is shown in S5D Fig. (G) Working model. Upper panel. FA mouse embryos depend on the canonical TGFβ-SMAD3 pathway for activating the expression of DNA-PKcs and perform NHEJ thus allowing Fancd2^-/- embryo survival in absence of a functional FA pathway. Lower panel. Absence of the FA pathway and the canonical SMAD3 pathway is embryonic lethal, however a small fraction of Smad3 Fancd2-129B6F2 embryos activate the non-canonical ERK pathway thus circumventing embryonic death. In both cases surviving mice present with defective hematopoietic stem cells in the bone marrow. (H) Schematics showing that the TGFβ ligands and TGFβ receptors can engage the canonical (SMAD3) or the non-canonical (ERK1/2) pathways and activate expression of NHEJ genes. Upon inhibition of the canonical TGFβ pathway, activation of the non-canonical ERK pathway occurs. This possess the potential emergence of resistance to TGFβ pathway inhibitors or refractory to treatment patients, therefore combining TGFβ pathway inhibitors, to avoid the emergence of resistance, has to be considered for designment of future clinical trials. Data in (B), (C) and (E) are represented as boxplots. Data in (F) are represented as mean ± SEM. p values of 0.01 to 0.05 were considered significant (*), p values of 0.001 to 0.01 were considered very significant (**) and p values of < 0.001 were considered extremely significant (***, ****). See also S5 Fig.

https://doi.org/10.1371/journal.pgen.1010459.g005

Additional validation of these findings with immunofluorescence microscopy revealed a marked reduction of γH2AX foci in Fancd2^-/- and Smad3 Fancd2-129B6F2 cells exposed to the MEKi (Fig 5B) and a reduced number of DNA-PKcs foci (Fig 5C) and Fig 5D) and 53BP1 foci (Fig 5E), especially in Smad3 Fancd2-129B6F2 cells.

The MEKi rescued the Fancd2^-/- and Smad3 Fancd2-129B6F2 cells from the toxicity induced by MMC (Fig 5F). Finally, treatment with LSN3301240, an upstream inhibitor of the TGFβ pathway which blocks TGFβ RI, resulted in a reduction of DNA-PKcs levels in both Fancd2^-/- and Smad3 Fancd2-129B6F2 cell lines, confirming that the TGFβ pathway is active in both cell lines (S4D and S5B Figs).

We conclude that during embryogenesis, Fancd2^-/- embryos activate the canonical TGFβ-SMAD3 signaling pathway and thereby upregulate the expression of NHEJ genes, allowing survival in the absence of a functional FA pathway. This NHEJ upregulation is detrimental to adult hematopoiesis later in life (Fig 5G, upper panel). On the other hand, if FA embryos lose the canonical TGFβ pathway, due to Smad3 inactivation, the viable Smad3 Fancd2-129B6F2 embryos will activate alternative components of the TGFβ superfamily for survival. These rare surviving embryos are the exceptions to the synthetic embryonic lethality that prevails upon simultaneous loss of Fancd2 and Smad3 (Fig 5G, lower panel).

Limitations of study

Our study has limitations. Having studied more than one FA KO mice, such as Fanca^-/- mice, would have been useful to show that our results apply to more than one FA genotype, however the Fancd2^-/- mouse model recapitulates all the hematopoietic phenotypes exhibited by other FA mice [31, 32].

Exploring additional gestational days would give more information on how embryonic development advances in these DNA repair deficient mice, however DKO embryos are no longer observable on day E14.5 and having objective comparisons on the four genotypes of interest (WT, Smad3^-/-, Fancd2^-/- and DKO) was not possible for the present report. However, Fancd2^-/- embryos are viable and studying their full gestation will be of interest.

We did not discover a mechanistic connection between the levels of Bax and TGFβ pathway activity, however we observed that Smad3 Fancd2-129B6F2 embryos, despite having the same genotype, have distinct gene expression profiles. Interestingly the levels of Bax expression correlated with the expression of NHEJ genes, i.e. lower levels of NHEJ correlated with high levels of Bax. This correlation made us hypothesize that those embryos with low NHEJ expression will not survive since Bax is a p53 target gene that promotes apoptosis [33]. We also believe that those Smad3 Fancd2-129B6F2 embryos that managed to survive were able to rewire their gene expression network and gain NHEJ expression through the non-canonical ERK, as observed in all the Smad3 Fancd2-129B6F2 weaned pups.

Ethics statement

All mice at Dana Farber Cancer Institute were housed according to the Institutional IACUC protocols and maintained four per cage, fed standard laboratory chow, and maintained on deionized sterilized water. All experimental procedures were approved by the Animal Care and Use Committee of the Dana Farber Cancer Institute. Mice were euthanized by CO₂ asphyxiation or by isoflurane overdose followed by cervical dislocation at the experimental time point or when loss of body weight, diarrhea, progressive dermatitis and any condition interfering with eating or drinking appeared. All mice at the University of Pittsburgh were housed at the Hlllman Cancer Center in a AAAA approved animal facility under the supervision of the Division of Laboratory Animal Research. All animal protocols were approved by the University of Pittsburgh Institute of Animal Control and Use Committee. All mice were euthanized by CO₂ inhalation followed by cervical dislocation at timepoints defined by the IACUC approved protocol or when showing signs of pain or distress such as loss of appetite, lacking of drinking, loss of 20% of their body weight, hunching of back, ruffling of fur or lethargy.

Mice

Fancd2^-/- (C57BL/6) [32] and Fancd2^-/- (129/Sv) (23) mice were bred in an established colony [34–36]. Smad3^-/- (C57BL/6) [37] and Smad3^-/- (129/Sv) [38] mice were bred from heterozygote breeding pairs obtained from Dr. Katherine Flanders, NIH, and Jackson Laboratories (Bar Harbor, Maine) respectively. The mice were genotyped by isolating DNA from tail clippings and performing PCR using primers described in the following paragraphs. Control mouse strains of C57BL/6, 129/Sv and 129/Sv X C57BL/6 F1 mice were maintained in cages at 37°C with water and food offered ad libitum.

Fancd2^-/- mice and Smad3^-/- mice on both C57BL/6 and 129/Sv backgrounds are infertile. To obtain DKO mice, heterozygous Fancd2^+/- and Smad3^+/- mice were bred. Smad3 Fancd2-129B6F2 (Smad3^-/- (129/Sv) Fancd2^-/- (B6)) mice were obtained by breeding Smad3^+/- (129/Sv) mice with Fancd2^+/- (C57BL/6) mice. Smad3 Fancd2-129129F2 mice were obtained by breeding Smad3^+/- (129/Sv) with Fancd2^+/- (129/Sv) mice. Smad3 Fancd2-B6B6F2 mice were obtained by breeding Smad3^+/- (C57BL/6) mice with Fancd2^+/- (C57BL/6) mice. Smad3 Fancd2-B6129F2 mice were obtained by breeding Smad3^+/- (C57BL/6) mice with Fancd2^+/- (129/Sv) mice (See also S2A Fig). The genotypes of newborns were identified by isolating DNA from the tip of the tail of weaned pups and performing PCR using primers specific for the Smad3 transgene, mutated Smad3 transgene, and Fancd2 transgene or mutated Fancd2 transgene.

Pregnant mice from the Smad3 Fancd2-129B6F2 breeding were sacrificed at day E12.5 and E14.5, the uterine horns removed, and the liver and placenta were dissected from the embryos. Embryos were genotyped by isolating DNA from the tip of the tail. Total RNA was extracted from the whole embryo, the fetal liver was used for protein extraction and western blot, a mixture of embryo and placenta was used for whole exome sequencing.

The primers for genotyping were 129/Sv Smad3 primers: wild type forward: 5’-TGA GTT TGC CTT CAA CAT GA; common reverse: 5’-CAC TCT GCC CAG TCC AAA G; mutant forward oIMR1100: 5’-GCT ATC AGG ACA TAG CGT TGG; 129/Sv Fancd2 primers: forward MG968: 5’-TCA GCC TCA CAT GGA GTT TAA CG; Common reverse MG1007: 5’-AAT TCG CCA ATG ACA AGA CGC; mutant forward MG1008: 5’-CAG GGA TGA AAG GGT CTT ACG C; C57BL/6 Smad3 primers: forward Smad3-1: 5′-CCA CTT CAT TGC CAT ATG CCC TG; reverse Smad3-2: 5′-CCC GAA CAG TTG GAT TCA CAC A; mutant forward pLoxpneo 5′-CCA GAC TGC CTT GGG AAA AGC; C57BL/6 Fancd2 Primers: forward OST2c: 5’-CAT GCA TAT AGG AAC CCG AAG G; reverse OST2a: 5’-CAG GAC CTT TGG AGA AGC AG; mutant forward LTR2b: 5’-GGC GTT ACT TAA GCT AGC TTG).

Cell lines

Bone marrow stromal cell lines were established by trypsin ionization of the adherent layer of long-term bone marrow cultures at week 4 [35, 36]. Stromal cells (mesenchymal stem cells) were grown in McCoy’s medium supplemented with 10% fetal calf serum and antibiotics and passaged weekly. Clonal lines were established by making the stromal cells into single cell suspension and using flow cytometry to sort single cells into individual wells of a 96 well plate. Once the cells have grown to confluence in the 96 well plates, they are expanded and made into a clonal cell line.

Isolation of Lin^- and LT-HSC from mouse

Mice were sacrificed and the bone marrow from tibia and femurs was harvested by gentle flushing with HBSS++ buffer [Hanks balanced salt solution (10-547F, Lonza) + HEPES (BP299-100, Fisher Scientific) + Fetal bovine serum (F2442, Sigma) + penicillin-streptomycin (15140–122, GIBCO)]. A 70 μM filter was used for filtering the samples and obtaining a single cell suspension. Lin^- enrichment was performed with lineage negative selection using the lineage cell depletion kit (130-090-858, Miltenyi).

For isolation of LT-HSCs, identified as Lin^-Sca-1⁺c-kit⁺ CD150⁺CD48^-, Lin^- cells were incubated with a mixture of biotin-labeled lineage antibody cocktail against CD3, CD11b, CD19, B220, Gr-1 and Ter119 (51-09082J, BD Pharmingen), and fluorochrome conjugate antibodies PE-Cy7-Sca (Clone D7, 558162, BD Biosciences), APC-c-kit (Clone ACK2, 135108, BD Biosciences), Pacific Blue-CD150 (Clone TC15-12F12.2, 115924, Biolegend) and APC-Cy7-CD48 (Clone HM48-1, 47-0481-82, e-Bioscience), followed by incubation with streptavidin-PE secondary antibody (554061, BD). LT-HSCs were sorted with a BD FACSAria cell sorter.

Alkaline comet assay

The alkaline comet assay was performed with the sorted LT-HSCs using the CometAssay kit (4250-050-K, Trevigen). LT-HSCs were mixed with low-melting-temperature agarose, coated on slides, and incubated in lysis solution overnight at 4°C. Next day cells were incubated during 1 h in unwinding NaOH solution and subjected to a current voltage of 12 V during 30 in a NaOH electrophoresis solution. Slides were dehydrated in ethanol solutions, washed, and stained with SYBRGreen (S7567, Invitrogen). Some cells were exposed to 10 Gy of irradiation using a 137Cs radiation source (model RS2000, Rad source) with a dose rate of 1 Gy/min and used as a positive control for DNA damage. Pictures were taken using a Zeiss Imager Z1 fluorescence microscope. Tail-length analysis was performed using the OpenComet plugin in the Image J software.

Colony forming unit assay

Clonogenic potential of mouse HSPCs was assessed in CFU assays by plating 20,000 Lin^- cells per triplicate in Methocult GF M3434 methylcellulose (03444, Stem Cell Technologies) and cultured for 7 days along with the TGFβ inhibitors Galunisertib or LSN3301240 at 200 nM, 500 nM and 5 μM final concentrations. The mouse hematopoietic colonies were scored after 7 days of culture at 37°C and 5% CO₂. Clonogenic potential of human HSPC was assessed in CFU assays by plating 3000 CD34⁺ cells per triplicate in human methylcellulose MethoCult H4434 Classic (04434, StemCell Technologies). Human colonies were quantified and classified after 14 days of culture at 37°C and 5% CO₂. Pictures were taken with the STEMvision System (StemCell Technologies).

Isolation of human CD34⁺ cells and viral transduction

Fresh healthy bone marrow samples were purchased from Lonza (1M-105, Lonza). Whole bone marrow was incubated with ammonium chloride (07800, StemCell Technologies) for 10 min on ice for lysis of red blood cells followed by a wash with PBS until obtaining a pellet of white cells. CD34⁺ cells were enriched using the Miltenyi kit (19056, Miltenyi Biotech) and following the manufacturer’s instructions.

Isolated human CD34⁺ cells were cultured in non-tissue culture treated plates for 36 hours in StemSpan SFEMII medium (09655, Stem Cell Technologies) with 100ng/ml of the following recombinant human cytokines: SCF (300–07, Peprotech), TPO (300–18, Peprotech), Flt3 (300–19, Peprotech) and IL-6 (200–06, Peprotech). Cells were then transferred to non-TC 96-well plates in a viral prep consisting of fresh media supplemented with polybrene (TR-1003-G, Sigma) and a lentivirus producing a shRNA against human FANCD2; a MOI of 50 was used for the scrambled shRNA and a MOI of 100 was used for the FANCD2 shRNAs. Plates were spun down at 2300 rpm for 30 minutes at RT and incubated for 12–16 hours. Selection media with 1 μg/ml puromycin (MIR 5940, MirusBio) was added to cultures 12–24 hours after viral infection. FA-like cells were selected in this puromycin-containing media for 72 hours.

Continuous bone marrow cultures and quantification of TGFβ in supernatant

The contents of a femur and tibia of 6-8-week-old mice of each genotype were flushed into 40 cm square plastic flasks in Fisher’s medium supplemented with 25% fetal calf serum and 10⁻⁶ M Hydrocortisone hemi-succinate [35, 36, 39]. Adherent layers were established by 4 weeks, and cultures fed by demi-depopulation of non-adherent cells and replaced with an equal volume of fresh medium. Cultures were maintained in a CO₂ incubator and assayed weekly for 35 weeks for number of cobblestone islands, production of nonadherent cells, percent confluency, and day 7 and 14 colony formation [39].

On weeks 9 and 10, cell culture media was obtained from the different cultures, and a TGF-B ELISA (Abcam, Cat. Ab119557) was performed.

Competitive repopulation assay

Competitive repopulation studies were performed for Smad3 Fancd2-129B6F2 mice (129/Sv Smad3^-/- C57BL/6 Fancd2^-/-) and Smad3 Fancd2-129129F2 mice (Smad3^-/- Fancd2^-/- all on 129/Sv background) [40]. Bone marrow from Smad3 Fancd2-129B6F2, 129/Sv Smad3^-/-, or C57BL/6 Fancd2^-/- mice were compared to C57BL/6 bone marrow by isolating bone marrow from male and female mice and mixing with bone marrow from C57BL/6 female and male mice, respectively, at ratios ranging from all male bone marrow to all female bone marrow. For Smad3 Fancd2-129129F2 experiments, bone marrow from male and female 129/Sv Fancd2^-/-, 129/Sv Smad3^-/-, and Smad3 Fancd2-129129F2 mice was mixed with bone marrow from female and male 129/Sv bone marrow, respectively.

The mixtures were injected intravenously (IV) into C57BL/6 (A) or 129/Sv (B) mice, which had received 10 Gy total body irradiation [41]. Bone marrow from 4–6-week-old mice of each genotype, and of each gender, was prepared as single cell suspensions and injected IV at 10⁶ cells per 10 Gy total body irradiated (TBI) mice, (JL Sheperd Model 68 Cesium irradiator, JL Sheperd and Associates) 300 cGy/min, recipient mice opposite gender in ratios of 1:0, 1:10, 2.5:7.5, 1:1, 7.5:2.5, 10:1 or 0:1 [40]. Groups of recipient wild type mice received IV injection of fresh bone marrow 24 hrs after TBI. Transplanted recipient mice were maintained for 120 days, and then bone marrow harvested and assayed for the relative contribution of donor bone marrow from each source by quantitation of Y-chromosome specific DNA [40]. At 120 days after bone marrow injections, the mice were sacrificed, bone marrow isolated, DNA extracted, and PCR performed with primers specific for the Y chromosome. The percent of mice negative for the Y chromosome (Percent Negative for Reconstitution) was plotted against the number of male cells injected.

Marrow stem cell numbers by flow analysis

Triplicate marrow samples from each mouse were analyzed by single cell, 7-color flow analysis of 1 x 10⁷ cells [40]. Bone marrow was made into single suspensions and incubated with the following antibodies to PE-eFluor 610-CD45, FITC-CD3e, FITC-CD5, FITC-CD8a, FITC-B220, FITC-GR-1, FITC-TER119, FITC-CD41, APC-eFluor 780-CD48, PE-Sca-1, PE-Ch7, and APC-CD150 for one hour. The cells were washed three times with PBS and analyzed by flow cytometry for bone marrow stem cells.

Protein extraction and western blotting

Whole cell lysates were prepared using RIPA cell lysis buffer (9803, Cell Signaling) and 1 mM PMSF (8553s, Cell Signaling). Lin^- cells, cells were cultured for 24 h in StemSpan SFEM medium (09600, StemCell Technologies) containing 2% L-glutamine (25030-081GIBCO), 1% penicillin/streptomycin (15140–122, GIBCO), 100 ng/ml SCF (250-03-10UG, Peprotech) and 100 ng/ml TPO (315-14-10UG, Peprotech). Lin^- cells were exposed to TGFβ1 (5 ng/ml) or TGFβ3 (5 ng/ml) along with LSN3301240 (5 μM) during 2 h. Western blots were performed using the following antibodies SMAD2/3 (86855, Cell Signalling), phospho-SMAD2 (ab3849, Millipore), phospho-SMAD2/3 (8828s, Cell Signaling) and Vinculin (sc-25336, Santa Cruz) antibodies.

Bone marrow stromal cell lines were exposed to irradiation (5 or 10 Gy), MEK inhibitor PD0325901 (10 μM) or TGFβ inhibitor LSN3301240 (5 μM). Western blots were performed with the following antibodies DNA-PKcs, 53BP1, RAD51, phospho-SMAD3 (9502s, Cell Signaling Technology, Danvers, MA, USA), p-ERK and actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Antibodies for analysis of fetal liver were DNA-PKcs, 53BP1, pERK1/2, total ERK1/2, CD45 CD41 and Vinculin.

DNA damage sensitivity assay

For Mitomycin C sensitivity assay, mitomycin C was added daily to Smad3 Fancd2-129B6F2, Smad3 Fancd2-129129F2, Smad3 Fancd2-B6B6F2 and Smad3 Fancd2-B6129F2 bone marrow stromal cell lines for 3 consecutive days at concentrations of 0, 5, 10, 15 or 20 ng/ml per day. The cells were then trypsinized and prepared as single cell suspensions, plated in each of 4 well plates at 500 cells per well and then incubated at 37°C in a CO₂ incubator for 7 days. The plates were stained with crystal violet and colonies of greater than 50 cells counted [35, 36]. For IR sensitivity assay, the Smad3 Fancd2-129B6F2 stromal cell line was exposed to 2.5, 5 and 10 Gy of IR, let to recover during 5 days and viability assessed with the CellTiter Glo viability assay.

RNA extraction and targeted RNA sequencing

RNA was extracted from embryos using the Midi RNA extraction kit (74034, QIAGEN). RNA quality was assessed using an Agilent Bioanalyzer and RNA Nano Chip in the Biopolymers Facility of Harvard Medical School. For gene expression analysis a QIAseq Targeted RNA panel targeted was designed (CRHS-10510Z-219-12, 333022, QIAGEN). Index assignment was done using the QIAseq Targeted RNA 96-index I kit (333117, QIAGEN). Libraries were sequenced in an Illumina NextSeq 500 Mid Output flow cell and run in an Illumina NextSeq 500 sequencer in the Biopolymers Facility of Harvard Medical School.

Gene expression analysis

Targeted RNAseq was performed using a panel of 203 mouse genes belonging to the DNA repair and TGF-β pathways. The panel with 9 reference genes were designed using the QIAseq Targeted RNA Panel with molecular barcode technology. Libraries were prepared according to manufacturer’s instructions. Next-generation sequencing was performed on the Illumina NextSeq 500 sequencer according to manufacturer’s instructions in the Biopolymers Facility at Harvard Medical School (https://www.genome.med.harvard.edu/).

Targeted RNAseq data were preprocessed as follows. First, QIAGEN’s GeneGlobe online portal was used to quantify gene expression counts from the number of unique molecular barcodes in the raw sequencing reads. Next, suitable reference genes were selected using geNorm [42] to be used for normalization. Finally, the gene expression counts were normalized using the selected reference genes and transformed into log2-counts per million (log2-CPM) values.

The relationship between the samples was examined using the Principal Component Analysis (PCA) plots, and the multi-dimensional scaling (MDS) plots which were generated by the plotMDS function of the limma R package [43]. PCA and MDS plots indicated the library preparation day could be a potential confounding factor.

Differential gene expression analyses were performed using the edgeR package [44], and the DESeq2 package [45], with the raw expression counts and the selected reference genes as inputs, and with library preparation day as a covariate. Both algorithms were used to have more confidence in the results. The estimated log-fold-changes of the two algorithms were confirmed to be highly similar. Multiple testing correction to control the false discovery rate (FDR) was performed using the Benjamini-Hochberg procedure on the p-values. The volcano plots of the edgeR results were generated using the ggplot2 package in R.

DNA extraction and whole exome sequencing

DNA from day E12.5 embryos and placentas were extracted using the DNA extraction kit from QIAGEN. DNA samples were submitted to GENEWIZ (South Plainfield, NJ) for library preparation and sequencing. Sequencing libraries were prepared with the HiseqX V2.5 sequencing kit and sequenced in a Illumina platform with a sequencing configuration of HiSeq, 2x150bp sequencing, single index. A total data output of ~350M raw paired-end reads were obtained per lane. FASTQ files were obtained for mutational signature extraction.

Whole exome sequence analysis

The paired-end whole exome sequence data were preprocessed following GATK Best Practices. Briefly, for each sample, the raw paired-end sequencing reads were mapped to the reference mouse genome C57BL/6J (GRCm38; mm10) using the Burrows-Wheeler aligner [46] (BWA-MEM; version 0.7.17). Duplicate reads were marked using Picard (version 2.19). The indels were realigned using GATK3 [47] (version 3.8). Finally, base quality scores were recalibrated using GATK4 (version 4.1).

Variant calling was performed using GATK3 UnifiedGenotyper (version 3.8) in the targeted coding regions. All 13 embryos were called together without down sampling. BCFtools [48] was then used to filter out some potentially low-quality variants with these parameters: “-g3 -G10 -e’QD<2.0 || FS>60.0 || SOR>5.0 || MQ<30’”. Only biallelic SNVs were selected for signature analysis. We discovered a total of over 128k SNVs from all 13 embryos.

We next identified the germline and somatic variants. One large source of known germline variants came from the 129S1 strain. We identified over 57k known 129S1 germline variants within the targeted regions, that were discovered from the whole genome sequencing efforts at The Wellcome Trust Sanger Institute [49]. Over 90% of the known 129S1 germline variants were rediscovered in the exomes of the embryos.

The scatter plots of the VAFs of the 129S1 germline variants were plotted using ggplot2 R package.

To identify other germline variants and other false variants, and somatic variants, we developed a simple procedure: if a variant was present in more than one embryo, it’s likely to be germline or other false variants; and if it was seen in only one embryo it is likely to be somatic. The exact procedure was as follows: if the highest variant allele fraction (VAF) was above 0.05, and the second highest VAF was more than 40% of the highest VAF, then it’s germline or false variant, else it’s a somatic variant. Variants with the highest VAF smaller than 0.05 were excluded. These thresholds were optimally picked so that most of the known 129S1 germline variants were identified. Upon manual visual inspection of VAF plots, we identified and removed several more clusters of variants that were likely germline, they appeared in clusters in only one embryo. Copy number analysis was performed following the GATK (version 4.1) workflow for detection of copy ratio alterations and allelic segments. The analysis-ready BAMs were used as inputs. The two wild type embryos were used to create the panel of normals (PoN) reference.

Immunofluorescence

Cells were grown on coverslips and fixed for 15 min in 4% paraformaldehyde (30525-89-4, Electron Microscopy Sciences) in PBS, then permeabilized with ice-cold methanol during 2 min at room temperature, washed immediately with PBS and incubated for 1 h in blocking buffer (10% Triton X-100 diluted in PBS with 10% normal goat serum.

Cells were incubated with primary antibodies anti- phospho-histone H2A.X (Ser 139) antibody (2577s, Cell signaling) anti-DNA-PKcs and anti-53BP1 diluted in IF buffer (1% BSA diluted in PBS and 10% Triton X-100 diluted in PBS) in a humidified chamber overnight at 4°C. The next day, cells were washed with PBSs threetimes and incubated for 1 h with secondary antibodies anti rabbit Alexa Fluor-488 (20E3, Cell Signaling) and Alexa Fluor- diluted 1:200 in IF buffer.

Slides were washed three times in PBS, counterstained and mounted with ProLong Gold antifade reagent with DAPI (P36931, Life Technologies). Images were taken with a Zeiss Imager fluorescent microscope.

Statistical analysis

Densitometry in western blots was analyzed with ImageJ software. Relative expression was calculated based on the intensity of each band over the intensity of its loading control. Data were normalized using WT levels or basal conditions as reference.

For analysis of frequency of births, we tested whether the frequency of births was significantly different from an expected frequency of 1 in 16, using a two-sided proportional test with p-values adjusted for multiple tests using the Bonferroni method. For competitive repopulation assay analysis, a single-hit model was fitted for each cell type, and comparison between the two cell types was done with the likelihood ratio test using the asymptotic chi-square approximation followed by the Bonferroni test.

For in vitro assays, we used the D’Agostino-Pearson, Shapiro-Wilk and Kolmogorov Smirnoff tests when required for assessing normality. We identified outliers with the ROUT method. 2-way ANOVA and Dunn’s multiple comparisons test were used for comparing experimental groups. Two-tailed p values for statistical analysis were obtained using Student’s t test.

p values of 0.01 to 0.05 were considered significant (*), p values of 0.001 to 0.01 were considered very significant (**) and p values of < 0.001 were considered extremely significant (***, ****). Graphpad Prism 8 and the Elda function in statmod package of R were used for statistical analysis.