METHOD OF GENERATING 2 CELL-LIKE STEM CELLS
20200131473 ยท 2020-04-30
Inventors
Cpc classification
C12N2501/999
CHEMISTRY; METALLURGY
C12N5/0606
CHEMISTRY; METALLURGY
C12N2506/45
CHEMISTRY; METALLURGY
C12N2740/15043
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to 2 cell-like stem cells and particularly, although not exclusively, the generation of such cells from pluripotent stem cells.
Claims
1. A method for inducing the expression of one or more 2 cell-like genes in a pluripotent stem cell, the method comprising activating replication stress response in a pluripotent stem cell.
2. The method of claim 1, comprising inducing replication stress in the pluripotent stem cell.
3. The method of claim 2, comprising exposing the pluripotent stem cell to one or more DNA damage inducing agents.
4. The method of claim 1, comprising culturing the pluripotent stem cell in the presence of one or more agent selected from Aphidicolin, ATR, ETAA1, hydroxyurea or high O.sub.2 or exposing the cell to UV radiation or ionising radiation.
5. A method for inducing the expression of one or more 2 cell-like genes in a pluripotent stem cell, the method comprising activating the ATR pathway in the pluripotent stem cell.
6. The method of claim 1 or claim 5, comprising overexpressing ETAA1 or an ATR binding fragment thereof in the pluripotent stem cell.
7. The method of claim 6, wherein the pluripotent stem cell is infected with a lentivirus that expresses ETAA1 or an ATR binding fragment thereof.
8. The method of any one of the preceding claims wherein the one or more 2 cell-like genes is selected from the group comprising Eif1a-like genes (Gm5662, Gm2022, Gm4027, Gm2016, and Gm8300), MERVL, MT2_Mm, Dux, Gm4981, Zfp352, Zfp750, Tdpoz genes (Tdpozl and Tdpoz3), and Tmem92, Zscan4 genes (Zscan4a-Zscan4d), Dux, Tcstv3, GM12794, H2AX, P-CHK1, GM4340, Gm12794, Afp352, Sfp750, Tdpoz, Eif1a and Tmem19.
9. The method of any one of the preceding claims wherein the cell expressing one or more 2 cell-like genes is substantially totipotent.
10. The method of any one of the preceding claims wherein the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell.
11. The method of any one of the preceding claims, further comprising a step of differentiating the cell into an embryonic or extra-embryonic tissue cell.
12. The method of any one of the preceding claims, further comprising isolating a cell expressing one or more 2 cell-like genes from pluripotent cells that do not express one or more 2 cell-like genes.
13. A 2 cell-like stem cell prepared by the method of any one of claims 1 to 12.
14. A stem cell that expresses one or more of Zscan4 genes, Tcstv3, GM12794, Eif, muERV-L, H2AX, P-CHK1, GM4340, Gm12794, Afp352, Sfp750, Tdpoz, Eif1a and Tmem19.
15. Stem cell culture medium comprising a DNA damage inducing agent, the DNA damage agent being optionally selected from Aphidicolin, hydroxyurea or high O.sub.2.
16. Use of a DNA damage inducing agent in the culture of stem cells.
17. Stem cell culture medium comprising an ATR activating agent.
18. Use of an ATR activating agent in the culture of stem cells.
19. A population of pluripotent stem cells wherein at least 6%, 7%, 8%, 9% or 10% of the stem cells express one or more 2C like genes.
20. A pluripotent stem cell containing a plasmid encoding ETAA1 or an ATR fragment thereof.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0148] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:
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EXAMPLES
Example 1: Materials & Methods
Cell Culture
[0163] ESCs were grown in feeder free culture condition either with or without 0.1% gelatin coat and incubated in 37 C. and 3% 02 tension. ESC medium (high glucose DMEM (DMEM MediaGlutaMAX-I, Gibco)), 15% ESC qualified FBS (HyClone Fetal Bovine Serum), 2 mM L-glutamine, 1/500 home-made leukemia inhibitory factor, 0.1 mM non-essential amino acids, 0.1 mM 2-bmercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 1 mM Sodium Pyruvate) supplemented with two inhibitors (2i from Axon medchem) that are PD 0325901; highly specific non-ATP-competitive inhibitor of MEK (aka MKK) 1/2 at final concentration of 1 uM and CHIR 99021; specific glycogen synthase kinase GSK-3 inhibitor at final concentration of 3 uM. MEF cells were cultured in MEF medium (high glucose DMEM (Lonza), 10% north America FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 units/ml penicillin, 1 mM Sodium Pyruvate and 50 mg/ml streptomycin).
Treatments
[0164] All treatments including APH, HU, UV, ATRi, ATMi and Chk1i were added in to fresh ESC medium and the cells were kept in 37 C. and 3% 02 tension for overnight (16-17 hrs) unless mentioned in each particular result section. APH was used at various concentrations ranging from 0.3 uM to 6 uM that is mentioned in each experiment in order to induce mild to acute replication stress response. Similarly, HU was used at 0.1 mM-3 mM. ATMi (KU-55933) and ATRi (VE 822) were used at the concentration of 10 uM and 1 uM respectively. Moreover, Chk1i (UCN-01) at the concentration of 100 nM was used. UV radiation was performed at the dosage ranging from 2 to 10 J/m2. For induction of ETAA1-AAD expression, Dox was used at the concentration of 1 g/mL for 48 hrs.
Flow Cytometry (FACS)
[0165] ESCs were fixed and permeabilized using Cytoperm/Cytofix kit (BD Biosciences), and subsequently stained for one hour at room temperature with Alexa Fluor 647 anti-H2AX-Phosphorylated (Ser139) antibody (Biolegend) or P-CHK1-PE conjugated antibody (cell signaling 12268). Cells were washed and acquired on a FACS Calibur instrument or Attune NxT (Thermo Fisher Scientific). For Emerald-GFP acquisition, cells were trypsinized, collected and subsequently acquired without fixation. Cell cycle profiles were acquired after incubation with DAPI (4,6-diamidino-2-phenylindole) overnight. For Caspase3 FACS analysis after fixation and permeabilization, Cleaved Caspase3 antibody were used for one hr at RT followed by washes and one hr incubation in donkey anti rabbit secondary antibody. For Zscan4-Emerald-GFP acquisition cells were trypsinized, collected and subsequently acquired without fixation step.
RNA Extraction, cDNA Synthesis and qPCR
[0166] RNA was extracted using RNeasy mini kit (QIAGEN), quantified by NanoDrop spectrophotometer and cDNA was prepared from 2 g total DNA-depleted RNA using SuperScript III reverse transcriptase kit (Invitrogen cat #18080-093) following manufacturer's instructions. qPCR assay performed based on standard protocol using 10 SsoFast EvaGreen Supermix or later 1 LightCycler 480 SYBR Green I Master (Roche, 04707516001), 0.5 M primer mix and 5 ng of cDNA. GAPDH was used as internal control to normalise the qPCR data following C.sub.t method, 10 M primer mix and a total amount of cDNA corresponding to 5 ng of starting RNA for each reaction.
RNA Isolation from Embryos and cDNA Preparation
[0167] Cryopreserved C57BL/6N morulae purchased from Janvier Labs (QUICKBLASTO), were thawed according to the manufacturer's protocol. After recovery, a group of healthy morulae were treated with APH at final concentration of 1.5 M in KSOM culture medium for 4 hrs while the rest were kept as control. The day after, the same number of morphologically healthy and synchronized blastocysts (22 and 7 blastocysts in the 1.sup.st and 2.sup.nd round, respectively) were selected from both groups. The APH concentration and the treatment interval had no effect on the viability of the embryos. RNA isolation was performed based on the previously published works.sup.14,15. Briefly, following the removal of zona pellucida in acidic tyrode's solution (Sigma-Aldrich, T1788), the pool of embryos was collected in 10 l of lysis buffer containing 5 First Strand Buffer (from SuperScript III reverse transcriptase kit, ThermoFisher) supplemented with 0.1% Tween-20. Subsequently, embryos were mechanically ruptured by three cycles of freeze/thaw. As the quantity of isolated RNA is not detectable by NanoDrop spectrophotometer, the supernatant was directly used for cDNA preparation following centrifugation at 10621 g for 2 minutes. cDNA preparation and qPCR were performed following the aforementioned protocols.
Immunocytochemistry
[0168] Briefly cells fixed in 4% PFA and subsequently blocked for one hour in FBS (10%) and Triton (0.1%). Then, cells were incubated with primary antibody at 4 C. overnight, followed by washes and incubation with secondary antibody for an hour at room temperature. Next samples were mounted and images were acquired with wide field florescent microscope. Antibodies used in this study were Anti Oct-3/4 (Santa Cruz sc-5279), Anti Nanog (Abcam ab80892) and Plet-1 antibody (Nordic MUbio MUB1512P) and), Anti-GFP (Abcam, ab5450). Acquired figures were analyzed with ImageJ software.
Immunoblotting
[0169] The cells were trypsinized and washed with PBS and then lysed in RIPA buffer plus protease/phosphatase inhibitor cocktail (cell signaling, #5872), incubating for 30 minutes on a rotating wheel at +4 C. Lysates were sonicated with a Bioruptor Sonication System (UCD200) at high power for 3 cycles of 30 seconds with one minute breaks. Lysates were centrifuged at 13000 rpm for 20-30 minutes and clear supernatants were transferred to new tubes. The proteins were quantified using Bio-Rad protein assay and following manufacturer's instructions. For the detection of each protein, 35 g of total protein extracts were loaded. Standard western blot was performed using following antibodies: ATR antibody (Cell Signaling 2790), Phoshpho-Chk1 antibody-S345 (Cell signaling 2348), Phoshpho-Chk1 antibody S317 (cell signaling 12302), Zscan4 antibody (Abcam ab4340), total Chk1 antibody (Santa Cruz 8408) and Chk2 antibody (Millipore 05-649), Anti-P53 (Cell Signalling, 2524S) and Anti-p-P53 (S15) (Cell Signalling, 12571S), Anti-Goat ALexa488 (Thermo Fisher Scientific, A-11055), Anti-Rabbit Cy3 (Jackson ImmunoResearch, 711-165-152), Anti-Mouse Alexa647 (Thermo Fisher Scientific, A-31571).
Target Preparation for Microarray
[0170] The quality of total RNA was first assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, Calif.). Biotin-labeled cDNA targets were synthesized starting from 150 ng of total RNA. Double stranded cDNA synthesis and related cRNA was performed with GeneChip WT Plus Kit (Affymetrix, Santa Clara, Calif.). With the same kit was synthesized the sense strand cDNA before to be fragmented and labeled. All steps of the labeling protocol were performed as suggested by Affymetrix. Each eukaryotic GeneChip probe array contains probe sets for several B. subtilis genes that are absent in the samples analyzed (lys, phe, thr, and dap). This Poly-A RNA Control Kit contains in vitro synthesized, polyadenylated transcripts for these B. subtilis genes that are pre-mixed at staggered concentrations to allow GeneChip probe array users to assess the overall success of the assay. Poly-A RNA Controls final concentration in each target are lys 1:100,000, phe 1: 50,000, thr 1:25,000 and dap 1:6,667.
DNA Strip Hybridization
[0171] Hybridization was performed using the GeneAtlas Hybridization, Wash and Stain Kit. It contains a mix for target dilution, DMSO at a final concentration of 10% and pre-mixed biotin-labeled control oligo B2 and bioB, bioC, bioD and Cre controls (Affymetrix cat #900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM respectively. Targets were diluted in hybridization buffer at a concentration of 0.05 g/ul, denatured at 99 C. for 5 minutes, incubated at 45 C. for 5 minutes and centrifuged at 5,000 rpm for 1 minute. The Array Strip is then moved to a hybridization tray, which contains the hybridization cocktails. A single Gene 2.1 PEG Array Strip is hybridized with four different biotin-labeled targets. Hybridizations were performed for 20 hours at 48 C. in the GeneAtlas Hybridization Station. The Array Strips were washed and stained in the GeneAtlas Personal Fluidics Station. At the end of this procedure the array strips were placed into the imaging tray, which contains the Array Holding buffer.
Image Acquisition, Processing and Bioinformatics Analysis
[0172] The array strips were imaged using the GeneAtlas Imaging Station. Affymetrix GeneAtlas Command Console software was used to acquire the Array Strip images and generate.
Differential Expression Analysis
[0173] We imported the microarray CEL files into R-3.2.2 (R Core Team. 2015. R: A Language and Environment for Statistical Computing. https://www.r-project.org/ (Accessed Sep. 12, 2016) and normalized intensity values with RMA 26 normalization. We performed differential expression analysis using the limma 27 package, weighting arrays with the arrayWeights function 28 and calculating p-values with an empirical Bayes method 29.
[0174] Expression Z-scores were calculated using the probe set level RMA normalized microarray intensity values using the standard formula:
Z-score=(x)/
Where x is the RMA normalized microarray intensity, is the probeset level average intensity value across all samples, and is the probeset level standard deviation of the intensity values.
Gene Set Enrichment
[0175] The CAMERA gene set enrichment p-values were calculated using the limma package and the camera function 30. Gene sets were defined based on the MsigDB gene sets 31, using a mouse orthologous collection (Hu Y. 2016. Mouse and human orthologues of the MSigDB in R format. http://bioinf.wehi.edu.au/software/MSigDB/ (Accessed Sep. 12, 2016).
[0176] In the GSVA based enrichment analysis, we used the MsigDB gene sets, and transformed the individual gene expression values into gene set expression values with the GSVA package 32. We calculated the gene set differential expression as in the gene level differential expression analysis, only skipping the RMA normalization step.
Genotyping of ATR Seckel ESC
[0177] In order to obtain homozygous ATR Seckel ESC, all nine ESC lines derived from breeding of ATR Seckel heterozygous mice were genotyped using previously described primer sets 14. 300 bp and 545 bp bands correspond to WT and ATR Seckel / genotypes.
In-Vitro Differentiation Toward Giant Trophoblast-Like Cells
[0178] ATR Seckel and WT ESCs were seeded on gelatin coated 6-well plate in ESC medium and 24 hours post treatment, medium was changed to trophectoderm stem cell (TSC) differentiation medium, which contains: 30% RPMI 1640 (with 20% FBS, 1 mM pyruvate, 2 mM L-glutamine, 100 mM -mercaptoethanol), 70% of conditioned medium from mitomycin-C-inactivated fibroblasts, 25 ng/ml of FGF4 (R&D Systems, 235-F4-025) and 1 ug/ml of heparin (Sigma, H3149). We changed the medium with fresh one every other day to maintain TSCs. To induce giant cell differentiation established TSC were split at day 2 on gelatin coated plate. After 24 hrs medium was changed to RPMI 1640 (with 20% FBS, 1mMpyruvate, 2mML-glutamine, 100 mM -mercaptoethanol) in the absence of heparin and FGF4, therefore, we changed the medium every other day for 3 days.
Ex Vivo Differentiation
[0179] Cryopreserved C57BL/6NRj morulas purchased from Janvier lab (QUICKBLASTO), were thawed according to the manufacturer's protocol. After recovery, half of morphologically healthy and synchronized morulas were treated with 1.50 M aphidicolin for four hours and the other half were kept as an untreated control (about 20 embryos in each condition). The day after, the same number of healthy blastocysts (E3.5) were collected from each group for RNA extraction, cDNA preparation and qPCR assay.
Mouse Chimaera Assay
[0180] Briefly, morula stage embryos were bought from Charles River laboratories. They were thawed few hours prior to injection and about five to eight ESC were injected into the perivitelline space of high quality morula-stage embryos using laser-assisted technique and glass micro-capillary tubes. Finally, embryos were cultured for 24 hours at 37 C, 5% CO2 to allow the blastocysts development prior to the microscopic analysis.
[0181] For ETAA-1 inducible cells, 8-cell morulae were obtained from superovulated C57BL/6 prepubescent females from Janvier Labs. Ten ESCs were injected into each morula after piercing their zona pellucida with beveled microinjection needle. Embryos were kept in M2 medium during the injection and afterwards in KSOM medium at 37 C. under 5% CO.sub.2. Morulae were either cultured for 48 hrs to study ESC contribution at late blastocyst stage (E4.5), or transferred to pseudopregnant females and later dissected at E7.5. Chimerism and ESC contribution to the various embryonic layers were assessed by emission of mCherry fluorescence signal.
[0182] For the APH-treated ESCs, frozen morula-stage embryos were purchased from Charles River and Janvier laboratories. Embryos were thawed 2 hrs prior to injection and 4 to 8 ESCs were transferred into the perivitelline space by laser-assisted microinjection method. Afterwards, embryos were cultured for 24 hrs at 37 C., 5% CO.sub.2 until blastocyst stage (E3.5).
Lentivirus Production
[0183] pLVX-EF1-IRES-mCherry vector was co-transfected with plasmids that are expressing POL, REV, TAT, and the vesicular stomatitis virus envelope glycoprotein (VSV-G) into 80% confluent 293T cells using calcium phosphate precipitation in the presence of 25 mM chloroquine. The supernatant of transfected cells were collected every 24 hrs for two days and concentrated using PEG-it virus precipitation solution and viral particles were re-suspended in DMEM and frozen in small aliquots in 80 C.
Animals
[0184] All mice used to generate ATR Seckel ESCs and MEF lines were bred and maintained under specific pathogen-free conditions. C57Bl/6J and 129P2/OlaHsd mice were purchased from Charles River Laboratories Harlan Italy (currently known as Envigo), respectively. Animals were kept in ventilated cages in standard 12 hrs light-dark cycle. The procedure was approved by the FIRC Institute of Molecular Oncology Institutional Animal Care and Use Committee and performed in compliance with Italian law (D.Igs. 26/2014 and previously D.Igs. 116/92), which enforces Dir. 2010/63/EU (Directive 2010/63/EU of the European Parliament and of the Council of 22 Sep. 2010, on the protection of animals used for scientific purposes).
ESCs Derivation from ATR Seckel and Chk1 Mice
[0185] To establish ATR.sup.Sec/Sec ESCs, ATR.sup.+/Sec heterozygous mice were crossed. Morulae were recovered and cultured overnight in KSOM medium (Millipore, MR-020P-5D) under mineral oil. Blastocysts were placed on MEFs feeder layer in ESC medium with 2i and LIF at 37 C. under 5% CO.sub.2. Upon ICM expansion, cells were passaged on feeder layer a to obtain the first stock. The cells were then characterized by genotyping. To establish Chk1.sup.+/ ESCs lines, embryos at morula stage were cultured overnight in KSOM supplemented with 2i. On the following day, blastocysts were plated individually in 96-well plate and cultured in N2B27 medium supplemented with 2i and LIF. After 6-7 days, the ICM outgrowth was disaggregated using Accutase. Clumps of cells were expanded every second day, or when the size of colonies reached to proper expansion level.
MEFs Generation
[0186] To produce MEFs, C57BL/6 and 129P2/OlaHsd mice were used. MEFs were prepared by mechanical disaggregation, trypsinization and seeding of embryos (E12-E13) in MEF medium after removal of the head, tail, limbs, and internal organs. Each trypsinized embryo was plated into a 10 cm dish. Once cells reached to 90% confluency (after 48 hrs), were frozen and stocked for the subsequent experiments.
Drop-Seq Single Cell mRNA Sequencing
[0187] ESCs from CNTL, APH (6 M) and APH-ATRi experimental conditions were resuspended in PBS-BSA and processed with a microfluidic device according to the DropSeq Laboratory Protocol V3.1 from McCarroll's Lab website [http://mccarrolllab.com/dropseq/l]. For each condition, 3 distinct aliquots of collected emulsion, each containing 4000 beads underwent reverse transcription (RT) and fragment library preparation using Illumina Nextera XT Library Prep Kit. A distinct barcode was used for each library to allow subsequent demultiplexing of sequencing reads. mRNA sequencing was performed using an Illumina HiSeq2000 instrument, library fragments were sequenced at 50 base pairs (bp) in PE mode. Sequencing reads were aligned to UCSC Mouse Reference Genome version mm10 using STAR (version 2.5.3a), and processed according to DropSeq Alignment Cookbook V1.2 to generate a digital expression matrix of STAMPs (cells) for each experimental condition, which was then furtherly analyzed in the RStudio environment (R version 3.3.3) using Seurat package V2.0 from Satija Lab [http://satijalab.org/seurat/]. Seven-hundred most expressed cells were selected in each condition; genes expressed in less than 3 cells and cells expressing less than 200 genes were pruned from the dataset. Data from CNTL and APH conditions (1399 cells) or CNTL, APH and APH-ATRi conditions (2096 cells) were merged to generate two datasets, which were log normalised and scaled. Expression mean and variance to mean ratio (Log VMR>0.5) were used to estimate data dispersion and select variable genes across the dataset. Respectively 2614 and 2768 genes (in the 2-conditions and 3-conditions datasets) were used in principal component analysis (PCA) to identify the appropriate number of components to include in data modeling. Top 15 components were selected to explain dataset complexity. A shared nearest neighbor (SNN) graph and smart local moving algorithm was used to perform cells clustering; t-SNE (t-stochastic neighbour embedding) analysis was used to reduce (PCA) dimensionality and generate plots of cells distribution.
RNA Sequencing Library Preparation and Sequencing
[0188] RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, Calif., USA) and RNA integrity was checked with RNA Screen Tape on Agilent 2200 TapeStation (Agilent Technologies, Palo Alto, Calif., USA). All RNA samples had a RIN score of 10. RNA sequencing library preparation was prepared using Ribo-Zero rRNA Removal Kit and TruSeq Stranded Total RNA library Prep kit following manufacturer's protocol (Illumina, Cat # RS-122-2101). Briefly, rRNA was depleted with Ribo-Zero rRNA Removal Kit. rRNA depleted RNAs were fragmented for 8 minutes at 94 C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. Sequencing libraries were validated using DNA Analysis Screen Tape on the Agilent 2200 TapeStation (Agilent Technologies, Palo Alto, Calif., USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, Calif.) as well as by quantitative PCR (Applied Biosystems, Carlsbad, Calif., USA).
[0189] The sequencing libraries were multiplexed and clustered on 1 lane of flowcell. After clustering, the flowcell was loaded on an Illumina HiSeq 4000 instrument according to manufacturer's instructions. The samples were sequenced using a 2150 Pair-End (PE) High Output configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS) on the HiSeq instrument. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.17. One mis-match was allowed for index sequence identification. On average 29 million reads were generated for a sample and average insert size ranged between 204-231 for the samples.
RNA Sequencing Data Processing
[0190] Transcript and gene level quantification was done with Kallisto (version 0.43.0).sup.16. The RefSeq NM and NR sequence collection as of 2016 Nov. 21, was used to build the transcriptome index. Kallisto was run with the -bias option to perform sequence based bias correction on each sample. Additionally, all reads were mapped with the STAR spliced aligner (version 2.5.2b).sup.17 to the GRCm38 (mm10) genome after trimming the reads to 100 bp with Trimmomatic (version 0.36).sup.18. Repeat expression was quantified by counting fragments overlapping with the repeat annotation from the Choi et al. 2017 paper, with using FeatureCounts from the subread package (version 1.5.2).sup.19. FeatureCounts was run with the p B C -primary options.
RNA Sequencing Gene Level Differential Expression
[0191] Read counts for each sample estimated by Kallisto were imported into the R statistical environment (version 3.2.2) using tximport (version 1.1.2).sup.20. The limma (version 3.24.15) package.sup.21 was used to test for differential expression between CNTL and APH or CNTL and APH+ATRi treatments after TMM normalization and voom transformation.sup.22. A linear model was fitted with the limma ImFit function and the moderated t-statistics was calculated with the eBayes function. Genes were defined as differentially expressed if they had FDR adjusted p-values <0.05 and |log.sub.2 fold change|>1.
[0192] The robust z-score for each gene in each sample was calculated using the median and mean absolute deviation (MAD) for each gene across samples, and standardizing expression with the following formula:
where x is the expression in TPM for a single gene in a given sample.
RNA Sequencing Repeat Analysis
[0193] Read counts for each sample based on the STAR alignment were imported into the R statistical environment (version 3.2.2) and summarized on the repeat subfamily level. edgeR.sup.23 was used to test for differential expression between CNTL and APH treatments, after TMM normalization. All repeat subfamilies were dropped where the count per million value did not reach 1 in at least 2 samples. A linear model was fitted with the edgeR glmFit function and a likelihood ratio p-value was calculated with the glmLRT function. Repeat subfamilies were defined as differentially expressed if they had FDR adjusted p-values <0.05 and |log 2 fold change|>1.
RNA Sequencing Gene Set Enrichment Analysis
[0194] Gene set enrichment was calculated using a mouse version of MSigDB available from the website of the bioinformatics group at the Walter and Eliza Hall Institute (Accessed Sep. 12, 2016). The CAMERA method.sup.24 available in the limma package was used to check for significant enrichment of specific gene sets between the CNTL and APH treatments, using the same TMM normalized read counts as in the gene differential expression analysis. Additionally, the GSVA.sup.25 package was used to transform gene level read counts per sample to a gene set level enrichment score per sample and for each MSigDB gene set. After this, the same limma analysis was carried out on the gene set enrichment values as for the gene level data. Gene sets were defined as differentially expressed if they had FDR adjusted p-values <0.05 and |log.sub.2 fold change|>0.5.
RNA Sequencing Literature Comparison
[0195] For all of the literature comparisons, the same transcript and gene level quantification methods and transcriptome annotations were used as for our data using the same limma package and methods to define differentially expressed genes between conditions. The Fisher-test was used to check for the significance of the overlaps in differentially expressed genes between our data and the literature datasets.
Transfection
[0196] ESCs were plated in 2i plus LIF medium as described above without antibiotics. Lipofectamine 2000 (Sigma-Aldrich) was diluted 1:50 in Opti-MEM (ThermoFisher) and incubated for 5 minutes at room temperature. Dux MISSION esiRNA pool (Sigma-Aldrich, EMU204411) or Trp53 siRNA (Sigma-Aldrich, SASI_Mm02_00310137) were diluted in Opti-MEM to the opportune concentration and then added to the lipofectamine emulsion at 1:1 ratio. This mixture was incubated for 20 minutes at room temperature and then was added to the cells suspension in culture medium. The final concentration of Dux and Trp53 were 75 nM and 40 nM, respectively. For each experiment a sample was transfected with MISSION siRNA universal negative control (scrambled siRNA) (Sigma-Aldrich, SIC005) at the same concentration used for the esiRNA or siRNA of interest. Gene expression was assessed 48 hrs post transfection.
ETAA1-AAD Cloning, Lentivirus Production and Induction
[0197] Lenti-XTM Tet-On 3G Inducible Expression System was used to express ETAA1-AAD protein in ESCs. The cDNA of ETAA1-AAD (kind gift from Niels lab) was cloned in pLVX-TRE3G vector. Viral particles of pLVX-TRE3G-ETAA1-AAD and pLVX-EF1a-Tet3G vectors were separately produced (as mentioned above). ESCs were co-infected with both lentiviruses in the presence of 8 g/mL polybrene, and subsequently infected cells were undergone puromycin and neomycin selection for two weeks. To induce ETAA1-AAD expression, selected ESCs were treated with 1 g/mL Dox for 48 hrs.
Example 2: RS Increases 2C-Like Cells in ESCs Culture and Activates 2C-Like Gene Expression in Mouse Embryos
[0198] Maintenance of genome stability despite unlimited self-renewal is a unique feature of ESCs.sup.26. Recent evidences have implicated that the key 2C-embryo-specific gene, Zscan4, is also expressed in ESCs where its transient burst was shown to play a critical role in maintaining genomic stability 5, 10. To evaluate the physiological relevance of these findings in vivo, we asked if forced induction of replication stress could activate the expression of key 2C-embryo specific genes in morula stage mouse embryos. To this aim, healthy synchronized morula-stage embryos were treated with polymerase inhibitor, aphidicolin and subjected to qPCR assay (
[0199] In spite of the eminent genome stability, the presence of replication stress has been well documented in mESCs 11 and has been linked to high basal level of histone variant H2AX phosphorylation (H2AX). As Zscan4 is periodically expressed in a fraction of ESCs, we tested whether its expression is associated with the accumulation of spontaneous replication stress. To test this hypothesis, we first examined the basal expression level of H2AX and P-CHK1 as the indicators of DNA replication stress response in Zscan4-Emerald ESCs. Interestingly, flow cytometry analysis revealed a significant enrichment of H2AX and P-CHK1 cells within Zscan4-Emerald positive ESC population with respect to Zscan4-Emerald negative ESC population (
[0200] Due to the important role of MuERV-L-derived LTR elements in shaping the transcriptional signature of 2C-stage embryos 12, we next asked whether replication stress induced Zscan4 expression was accompanied by activation of MuERV-L retroelements. To this end, we exposed ESCs to a wide range of replication stress inducing agents including aphidicolin (APH), hydroxyurea (HU) and ultraviolet light (UV) (
[0201] Two cell (2C)-like cells have been shown to reduce the expression of the pluripotency markers at the protein but not transcriptional level 1, 2. Thus, in order to understand if replication stress induced cells share such features with the 2C-like cells, we checked the expression of the canonical pluripotency markers upon APH treatment. In agreement with previous publications 1, 2, we found a mild downregulation of Nanog, Pou5f1 (Oct4) in Zscan4 positive cells upon immunostaining (
[0202] Finally, we found that APH treatment do not alter the expression of main 2C-specific markers such as MuERV-L in mouse embryonic fibroblast (MEF) lines with distinct genetic backgrounds suggesting that the activation of 2C-like genes upon replication stress could be cell type specific (
[0203] Next, to gain further insight into the mechanisms through which replication stress response could contribute to the reactivation of 2C-like specific genes in ESC culture, we asked if suppression of DNA damage response pathway (DDR) could restrain the reactivation of main 2C-like genes under normal culture condition. Thus, we inhibited DDR by addition of specific ATM and ATR inhibitors (KU-55933 and VE 822, respectively) in Zscan4-Emerald ES cells. Flow cytometry analysis revealed that the addition of ATRi could markedly reduce the number of Zscan4-Emerald positive cells however, treatment with ATM inhibitor did not change the number of the Zscan4 positive population in the dish significantly (
[0204] Next, we asked whether inhibition of DDR could revert the transcriptional activation of MuERV-L and Zscan4d under replication stress inducing condition to uncover which specific pathway is mostly involved in replication stress induced 2C-like genes activation. To this aim, Zscan4-Emerald ES cells were treated with specific ATM and ATR inhibitors upon APH and HU treatment. While the immunoblot results confirmed the robust and highly specific inhibition of ATM and ATR pathway upon replication stress (shown by the phosphorylation status of CHK1, CHK2), flow cytometry analysis and immunoblot revealed that ATR inhibitor could robustly suppress the elevation of 2C-like specific genes whereas ATM inhibition could not alter the elevated expression of Zscan4 upon APH and HU treatment (
[0205] Next, to understand if activation of ATR dependent response could result in global transcriptional alterations similar to that of 2 cell-stage embryos, we performed whole gene expression profiling on two established mESC lines namely E14 and R1 upon APH treatment. Analysis of differentially expressed genes (DEGs) (False discovery rate (FDR)-adjusted P-value cutoff) identified 193 upregulated genes with more than two-fold changes in gene expression upon APH treatment, and only 21 downregulated genes which is in agreement with general openness of the chromatin in totipotent state 1 (
[0206] Next, we aimed to dissect the role of ATR on the global transcriptional reversion of replication stress induced 2C-specific genes. While the geneset enrichment analysis confirmed a robust silencing of DNA repair signaling pathway upon addition of specific ATR inhibitors and Caffeine (
[0207] To understand how ESCs coordinate these functions, we asked how ESCs respond to RS at the single cell level. To this end, we performed single cell transcriptional profiling.sup.27 on E14 mouse ESCs cultivated in Leukemia Inhibitory Factor (LIF) plus MEK and GSK inhibitors (2i) upon treatment with aphidicolin (APH), a reversible inhibitor of DNA polymerase that induces RS by stalling replication fork progression.sup.28. Unsupervised clustering analysis.sup.27 identified four distinct clusters of cells (
[0208] Due to the key role of Dux and MERVL-derived long terminal repeat (LTR) elements in shaping the transcriptional signature of 2C-stage embryos and 2C-like cells.sup.28-10,31, we next asked whether RS-induced expression of Zscan4 genes was accompanied by activation of these retroelements. The qPCR results revealed a linear correlation between concentration of RS inducing agents and the expression of key 2C-like markers, Dux and MERVL (
[0209] 2C-like cells reduce the expression of pluripotency markers at protein but not transcriptional level.sup.1,2,31. Thus, to understand whether RS-induced cells share such features with the 2C-like cells, we monitored the expression of the canonical pluripotency markers upon APH treatment. In agreement with previous findings.sup.1,31, we detected downregulation of NANOG and POU5F1 (also known as OCT4) proteins in Z.sup.+ cells by immunostaining (
[0210] The Z+/MERVL cells were reported to be present in all phases of the cell cycle albeit with higher percentage in G2/M phase.sup.13. Of note, the cell cycle analysis on pZscan4-Emerald ESCs confirmed that APH treatment neither at low (0.3 M) nor at high (6 M) concentrations could increase G2/M population in culture (
[0211] Overall, these findings indicate that RS leads to the activation of 2C embryo specific genes in ESCs and mouse embryos.
Example 3: Activation of Endogenous DNA Damage Pathways is Responsible for the Emergence of 2C-Like Cells Under Normal Culture Condition
[0212] Next, to gain further insight into the mechanisms through which RS response (RSR) could contribute to the emergence of 2C-like cells in ESCs culture, we asked whether activation of the endogenous DNA damage response pathways (DDR) is responsible for the emergence of 2C-like cells under normal culture condition. Thus, we inhibited DDR by treating pZscan4-Emerald ESCs with specific ATM and ATR inhibitors (ATMi, KU-55933 and ATRi, VE 822, respectively). FACS analysis revealed a marked reduction in the number of Em.sup.+ cells upon ATR inhibition. However, treatment with ATMi did not significantly reduce the fraction of Em.sup.+ cells in culture (
[0213] In agreement with our findings, single cell transcriptional profiling revealed that the number of RS-induced cells in the newly emerging cluster was significantly reduced upon ATR inhibition (
Example 4: ATR and CHK1-Mediated RS Response Triggers Activation of Key 2C-Like Genes in ESCs
[0214] Next, to exclude any unspecific inhibitory effect of ATR and CHK1 inhibitors on the transcriptional repression of 2C-like genes, we aimed to generate ATR Seckel and CHK1 heterozygous ESCs from previously reported mice models 14, 15 as the complete ablation of ATR and CHK1 causes embryonic lethality. To this aim, two heterozygous mouse for ATR Seckel and CHK1 allele were crossed and ESC were isolated at embryonic day 3.5 (E3.5). On the basis of genotyping results, two homozygous ATR Seckel and two heterozygous CHK1 mESC were used for further characterization (
[0215] Although immunoblot results confirmed that the level of ATR and CHK1 protein is severely reduced in comparison to the wild type control (
[0216] Importantly, the qPCR results in two ATR Seckel and two control lines on a large panel of 2C-like specific genes validated our previous data. Of note, similar to inhibitory impact of ATR inhibitors on expression of 2C-like genes in ESC, APH treatment on ATR Seckel ESC led only to a milder increase of 2C-like genes expression unlike normal wild type ESC (
[0217] To further validate these findings, we derived ATR-deficient Seckel (ATR.sup.Sec/Sec) and CHK1.sup.+/ ESCs from previously reported mouse models.sup.33,34 as the complete ablation of ATR or CHK1 causes embryonic lethality in mice.sup.34,35. To this end, ESC lines were established in culture from pre-implantation embryos obtained from crosses between heterozygous mice either for ATR Seckel or for CHK1 KO alleles. On the basis of genotyping results, two homozygous ATR Seckel and two heterozygous CHK1 ESC lines were characterized for further investigations (
[0218] Overall, these data demonstrate that an ATR and CHK1-stimulated response to endogenous and induced RS regulates the activation of 2C-like specific genes.
[0219] Recent evidence point to the activation of the ATR kinase by the RPA-binding protein, Ewing's tumor-associated antigen 1 (ETAA1) 16. Importantly, several lines of evidence suggested that, the H2AX positivity caused by ATR activating domain (ETAA1-AAD) overexpression is not a consequence of DNA breakage 16, 17. Hence, in order to exclude the possibility of the activation of 2C-like gene due to physical damage of DNA and not due to ATR activation, we aimed to activate ATR in DNA damage free context through ETAA1-ATR activating domain (AAD) overexpression. To this aim, Zscan4-Emeraled ESCs were infected by two independent ETAA1-ADD expressing lentiviruses. FACS analysis on mCherry positive infected cells, confirmed activation of ATR pathway (i.e., increase in the number of H2AX positive cell) upon infection with ETAA1-AAD lentivirus with respect to the empty vector (EV) infected cells (
[0220] These data collectively further consolidate that activation of ATR, even in the absence of replication stress, could trigger the global reactivation of 2C-like gene network and that ATR inhibition exerts a robust suppressive impact on such transcriptional activation.
Example 5: ETAA1-Mediated Direct Activation of ATR Induces 2C-Like Cells in a RS-Free Context
[0221] Recent evidence showed that ATR kinase can be directly activated by the RPA-binding protein, Ewing's tumor-associated antigen 1 (ETAA1) in the absence of RS.sup.38,39. Hence, to confirm that the activation of 2C-like genes is due to ATR-mediated response but not the physical damage to DNA, we aimed to activate ATR in a DNA damage-free context through overexpression of ETAA1-ATR activating domain (ETAA1-AAD) in ESCs. To this end, pZscan4-Emerald ESCs were infected with two independent lentiviruses generated from two different clones of ETAA1-expressing lentivectors in which ETAA1-ADD was expressed under the control of a doxycycline (Dox) inducible promoter, and subsequently selected against puromycin and neomycin for two weeks (
[0222] Importantly, several lines of evidence demonstrated that the H2AX positivity caused by the overexpression of ETAA1 was not a consequence of DNA breakage.sup.38,39. We also confirmed that ATR activation through ETAA1 expression leads to the phosphorylation of H2AX and CHK1 but not CHK2 in ESCs (
[0223] These results collectively indicate that DNA damage-free ETAA1-stimulated ATR activation in ESCs is sufficient to activate 2C-like genes through DUX induction.
[0224] Our results so far indicated that ATR activated cells exhibit similar transcriptional profile and characteristics of 2-cell-stage embryos. Strikingly, computational gene-sets analysis of APH vs CNTL through molecular signature database (MSigDB), identified a strong and highly significant enrichment in two placenta-related gene categories which are robustly suppressed upon ATR inhibition by specific ATR inhibitor and Caffeine (FC and p-value) (
[0225] In addition, whole transcriptional profiling results also revealed the activation of several key placenta related genes upon replication stress in ESC including; Dgka, Egfr, Slc7a8, Eng, Cyr61, Adm, Scn1b, Emp3, Anpep, Cryab and Hspb2, among which some (e.g., Dgka, Eng and Egfr) are interestingly involved in exosome formation and angiogenesis, the critical pathways that are potentially exploited by placenta to gain migratory, invasive and immune suppressive features 18 (
[0226] These findings led us to ask whether ATR activated ESC also gained expended development potency to be differentiated to extra-embryonic tissues. To test this hypothesis, both ATR Seckel and wild type ESC cells (upon treatment with APH) were differentiated in vitro toward trophoblast-like stem cells for three days followed by terminal differentiation to giant trophoblast-like cells upon withdrawal of FGF4 and heparin for 3 additional days (
[0227] The qPCR results for the giant trophoblast cell specific marker, Prl2c2 revealed the highest expression in ATR activated condition while ATR Seckel cells could not significantly upregulate such a marker in the absence of APH (
Example 6: ATR-Activated ESCs Gain Expanded Developmental Potential
[0228] Next, to understand if these cells have also gained distinct functional characteristics to give rise to extra-embryonic tissues along with inner cell mass derived lineages, FACS sorted positively pre-infected ESC with a lentivirus encoding mCherry protein were treated them with APH (
[0229] To test this hypothesis, both ATR.sup.Sec/Sec and ATR.sup.+/+ ESCs were cultivated in the absence or in the presence of APH and were subsequently differentiated in vitro toward trophoblast-like stem cells (TSCs) for three days, followed by terminal differentiation to TGCs upon withdrawal of fibroblast growth factor 4 (FGF4) and heparin for three additional days (
[0230] The qPCR results for the TGC specific marker, Pr/2c2, revealed the highest expression in ATR activated conditions, while ATR.sup.Sec/Sec cells could not significantly upregulate this gene in the presence of APH (
[0231] Next, to understand whether ATR-induced differentiation to TGCs is also mediated through transition to 2C-like state, we differentiated Dux WT and KO ESCs toward trophoblast cells upon ATR activation (
[0232] Next, to define the cell fate potential of ATR-activated ESCs during embryonic development, we traced the fate of their progenies in chimeric blastocysts. To this end, ten mCherry fluorescent protein-labeled (mCherry-labeled) Dox-iETAA1 ESCs were treated with Dox and subsequently microinjected into each C57BL/6N recipient mouse morulae to generate chimeric blastocysts. Untreated Dox-iETAA1 ESCs were injected in parallel as control. Next, the contribution of ESCs to inner cell mass (ICM) and trophectoderm (TE) of the blastocysts was monitored 48 hrs post-injection (
[0233] To further validate these findings, we then generated post-implantation chimeric embryos by microinjecting ten mCherry-labeled Dox-iETAA1 ESCs into each C57BL/6N morulae that were subsequently transferred to foster mothers. While WT ESCs contributed exclusively to the embryonic tissue (epiblast), ATR-activated cells contributed to both embryonic and extra-embryonic cell lineages in 50% (3 out of 6) of E7.5 (embryonic day 7.5) chimeric embryos (
Example 7: ATR Induces Transcriptional Signature of 2C-Like Cells in ESCs Through DUX Activation
[0234] Next, to understand whether activation of ATR-dependent response could result in global transcriptional activation of 2C-like genes, we performed high-throughput transcriptional profiling on three ESC lines, namely E14, R1 and MC1 upon APH treatment. Analysis of differentially expressed genes (DEGs) (FDR<0.05 and |log 2 fold change|>1) identified 3074 upregulated genes with more than two-fold change in gene expression upon APH treatment, and only 640 downregulated genes (
[0235] Considerably, qPCR results confirmed significant upregulation of main 2C-like genes upon APH treatment (
[0236] Recent reports demonstrated that 2C-like cells can be generated through genetic modulation of several factors including chromatin assembly factor-1 (CAF-1), KRAB (Kruppel-Associated Box Domain)-Associated Protein 1 (KAP1), microRNA-34 (miR-34) and DUX.sup.1,8,31,36. Thus, to test whether these factors were involved in ATR-induced expression of 2C-like genes, we compared the transcriptome of APH-induced ESCs with those published datasets. While we found a significant overlap with all the datasets, the highest number of overlapping genes was found with the transcriptome of CAF-1 KD ESCs (
[0237] Importantly, APH treatment could not induce the expression of key 2C-like genes in DUX KO ESCs, confirming that the expression of ATR-induced 2C-like genes requires DUX (
DISCUSSION
[0238] Here, we provide evidence that transition to bipotent 2C-like cells in ESCs culture is triggered by activation of ATR, a developmentally essential DDR gene, which plays a crucial role in the maintenance of stem cells both in embryonic and adult tissues.sup.35,41 First, we found a significant enrichment of H2AX.sup.+ and CHK1+ cells within Z.sup.+ cell population. Second, inhibition of RSR by specific ATR and CHK1 inhibitors could markedly reduce the number of 2C-like cells in ESCs culture. Third, ATR-deficient Seckel and haploinsufficient CHK1 ESCs exposed to RS could not significantly induce 2C-like specific genes. Finally, robust activation of ATR through ETAA1 overexpression could further increase the bipotent 2C-like cell population in ESCs culture in the absence of RS.
[0239] Importantly, we unraveled the mechanistic basis of ATR-stimulated transition to bipotent 2C-like cells by showing that this response is mediated by DUX, the key inducer of zygotic genome activation (ZGA) in placental mammals.sup.10,39. Although DNA damage induced cellular differentiation has been frequently shown in stem cells.sup.42,43, here we report for first time to our knowledge that RS in ESC leads to the transition to a more developmentally potent state that is required for the subsequent differentiation toward TGCs. Our results show that, in response to RS, ATR triggers Dux expression that in turn increases bipotent 2C-like cells in ESC culture through global transcriptional activation of 2C-like genes, including genome caretaker genes Zscan4 and Tcstv1/3.sup.5,10,12,39. In addition to their role in telomere elongation through activation of telomere sister chromatid exchange.sup.5,29, Zscan4 genes could also promote DNA repair by facilitating heterochromatin decondensation and DNA demethylation.sup.13,30,44. Therefore, ATR-mediated activation of Zscan4 genes likely contributes to promote ESCs genomic integrity in response to RS. Unexpectedly, our findings also show that ATR activation in ESCs can trigger the generation of ESCs with bidirectional cell fate potential (
[0240] ATR activation might also impact on cell fate at later stages of embryonic development. However, our findings clearly show that RS per se is not able to trigger 2C-like pathway in more committed embryonic cells such as MEFs, suggesting an involvement of robust repressive mechanisms in suppressing this pathway in differentiated cells. The consequence of unrepaired damage could be extremely serious during early embryonic development as it could subsequently result in embryonic lethality or transmission to the large populations of cells leading to teratogenicity or even germ lines incorporation. Therefore, it is vital for embryonic cells to respond efficiently to genotoxic stress through coordinated and integrated DNA damage repair pathways.
[0241] Though DNA damage induced cellular differentiation has been frequently reported in stem cells, to our knowledge, here for first time we report that replication stress in ESC, could lead to the transition to more primitive totipotent-like state followed by the global activation to 2C-like transcriptional network. More importantly, we found that the activation of 2C-like genes is mediated by ATR, which is reported to be essential to maintain stem cell self-renewal in embryonic and adult tissues. In addition to its involvement in DNA repair and response, ATR activation could boost the totipotency genes in face of exogenous and endogenous stresses. Upon failure of DNA repair, activation of trophectoderm differentiation program would prevent the contribution of ESCs with damaged DNA to developing tissues. It is tempting to speculate that activation of such ATR dependent pathway in committed adult stem cells might lead to expression of genes responsible for invasive and migratory behaviour of trophectoderm cells, which share key features with invasive and metastasizing cancer cells. This hypothesis would be compatible with replication stress being a major outcome of oncogene activity in cancer cells and would justify the potent suppressing role of ATR inhibitor in tumorigenesis.
[0242] In summary, our findings shed light on the endogenous and exogenous stimuli that could contribute to the cellular plasticity of ESCs and also provide a fundamental insight into the alternative mechanisms these cells exploit to respond to RS and maintain genome integrity.
REFERENCES
[0243] 1 Ishiuchi, T. et al. Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly. Nature Structural & Molecular Biology 22, 662-U635, doi:10.1038/nsmb.3066 (2015). [0244] 2 Macfarlan, T. S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57-+, doi:10.1038/nature11244 (2012). [0245] 3 Zhou, L. Q. & Dean, J. Reprogramming the genome to totipotency in mouse embryos. Trends Cell Biol 25, 82-91, doi:10.1016/j.tcb.2014.09.006 (2015). [0246] 4 Akiyama, T. et al. Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells. DNA Res, doi:10.1093/dnares/dsv013 (2015). [0247] 5 Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858-U866, doi:10.1038/nature08882 (2010). [0248] 6 Choi, Y. J. et al. Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells. Science 355, doi:10.1126/science.aag1927 (2017). [0249] 7 Amano, T. et al. Zscan4 restores the developmental potency of embryonic stem cells. Nature Communications 4, doi:Artn 1966 10.1038/Ncomms2966 (2013). [0250] 8 Hendrickson, P. G. et al. Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons. Nature Genetics 49, 925-+, doi:10.1038/ng.3844 (2017). [0251] 9 Whiddon, J. L., Langford, A. T., Wong, C. J., Zhong, J. W. & Tapscott, S. J. Conservation and innovation in the DUX4-family gene network. Nature Genetics 49, 935-+, doi:10.1038/ng.3846 (2017). [0252] De Iaco, A. et al. DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nature Genetics 49, 941-+, doi:10.1038/ng.3858 (2017). [0253] 11 Amano, T. et al. Correction of Down syndrome and Edwards syndrome aneuploidies in human cell cultures. DNA Research 22, 331-342, doi:10.1093/dnares/dsv016 (2015). [0254] 12 Zhang, Q. et al. Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells. Sci Rep 6, 19852, doi:10.1038/srep19852 (2016). [0255] 13 Eckersley-Maslin, M. A. et al. MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs. Cell Rep 17, 179-192, doi:10.1016/j.celrep.2016.08.087 (2016). [0256] 14 Falco, G. et al. Zscan4: A novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells. Developmental Biology 307, 539-550, doi: 10.1016/j.ydbio.2007.05.003 (2007). [0257] Jeong, Y. J. et al. Optimization of real time RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos. Molecular Reproduction and Development 71, 284-289, doi: 10.1002/mrd.20269 (2005). [0258] 16 Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nature Biotechnology 34, 525-527, doi:10.1038/nbt.3519 (2016). [0259] 17 Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21, doi:10.1093/bioinformatics/bts635 (2013). [0260] 18 Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120, doi: 10.1093/bioinformatics/btu170 (2014). [0261] 19 Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930, doi: 10.1093/bioinformatics/btt656 (2014). [0262] Soneson, C., Love, M. I. & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res 4, 1521, doi: 10.12688/f1000research.7563.2 (2015). [0263] 21 Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43, e47, doi:10.1093/nar/gkv007 (2015). [0264] 22 Law, C. W., Chen, Y. S., Shi, W. & Smyth, G. K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biology 15, doi:ARTN R29 10.1186/gb-2014-15-2-r29 (2014). [0265] 23 McCarthy, D. J., Chen, Y. S. & Smyth, G. K. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297, doi:10.1093/nar/gks042 (2012). [0266] 24 Wu, D. & Smyth, G. K. Camera: a competitive gene set test accounting for inter-gene correlation. Nucleic Acids Res 40, e133, doi:10.1093/nar/gks461 (2012). [0267] Hanzelmann, S., Castelo, R. & Guinney, J. GSVA: gene set variation analysis for microarray and RNA-Seq data. Bmc Bioinformatics 14, doi:Artn 7 10.1186/1471-2105-14-7 (2013). [0268] 26 Giachino, C., Orlando, L. & Turinetto, V. Maintenance of Genomic Stability in Mouse Embryonic Stem Cells: Relevance in Aging and Disease. International Journal of Molecular Sciences 14, 2617-2636, doi:10.3390/ijms14022617 (2013). [0269] 27 Macosko, E. Z. et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell 161, 1202-1214, doi:10.1016/j.cell.2015.05.002 (2015). [0270] 28 Aze, A., Sannino, V., Soffientini, P., Bachi, A. & Costanzo, V. Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression. Nature Cell Biology 18, 684-+, doi:10.1038/ncb3344 (2016). [0271] 29 Nakai-Futatsugi, Y. & Niwa, H. Zscan4 Is Activated after Telomere Shortening in Mouse Embryonic Stem Cells. Stem Cell Reports 6, 483-495, doi:10.1016/j.stemcr.2016.02.010 (2016). [0272] 30 Akiyama, T. et al. Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells. DNA Res 22, 307-318, doi:10.1093/dnares/dsv013 (2015). [0273] 31 Choi, Y. J. et al. Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells. Science 355, 596-+, doi:10.1126/science.aag1927 (2017). [0274] 32 Storm, M. P. et al. Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells. Plos One 9, doi:ARTN e89821 10.1371/journal.pone.0089821 (2014). [0275] 33 Murga, M. et al. A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging. Nature Genetics 41, 891-U846, doi:10.1038/ng.420 (2009). [0276] 34 Liu, Q. H. et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes & Development 14, 1448-1459 (2000). [0277] 35 Brown, E. J. & Baltimore, D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes & Development 14, 397-402 (2000). [0278] 36 Rowe, H. M. et al. KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463, 237-240, doi: 10.1038/nature08674 (2010). [0279] 37 Hoek, M. & Stillman, B. Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo. Proceedings of the National Academy of Sciences of the United States of America 100, 12183-12188, doi:10.1073/pnas.1635158100 (2003). [0280] 38 Bass, T. E. et al. ETAA1 acts at stalled replication forks to maintain genome integrity. Nature Cell Biology 18, 1185-+, doi:10.1038/ncb3415 (2016). [0281] 39 Haahr, P. et al. Activation of the ATR kinase by the RPA-binding protein ETAA1. Nat Cell Biol 18, 1196-1207, doi:10.1038/ncb3422 (2016). [0282] Abad, M. et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature 502, 340-+, doi:10.1038/nature12586 (2013). [0283] 41 Ruzankina, Y. et al. Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell 1, 113-126, doi:10.1016/j.stem.2007.03.002 (2007). [0284] 42 Schneider, L. et al. DNA Damage in Mammalian Neural Stem Cells Leads to Astrocytic Differentiation Mediated by BMP2 Signaling through JAK-STAT. Stem Cell Reports 1, 123-138, doi:10.1016/j.stemcr.2013.06.004 (2013). [0285] 43 Santos, M. A. et al. DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier. Nature 514, 107-+, doi:10.1038/nature13483 (2014). [0286] 44 Dan, J. M. et al. Zscan4 Inhibits Maintenance DNA Methylation to Facilitate Telomere Elongation in Mouse Embryonic Stem Cells. Cell Reports 20, 1936-1949, doi:10.1016/j.celrep.2017.07.070 (2017). [0287] Ferretti, C., Bruni, L., Dangles-Marie, V., Pecking, A. P. & Bellet, D. Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts. Human Reproduction Update 13, 121-141, doi:10.1093/humupd/dm1048 (2007). [0288] 46 Yasuda, T. et al. Recurrent DUX4 fusions in B cell acute lymphoblastic leukemia of adolescents and young adults (vol 48, pg 569, 2016). Nature Genetics 48, 1591-1591 (2016). [0289] 47 Yoshimoto, T. et al. CIC-DUX4 Induces Small Round Cell Sarcomas Distinct from Ewing Sarcoma. Cancer Research 77, 2927-2937, doi: 10.1158/0008-5472.Can-16-3351 (2017). [0290] 48 Macheret, M. & Halazonetis, T. D. DNA Replication Stress as a Hallmark of Cancer. Annual Review of Pathology: Mechanisms of Disease, Vol 10 10, 425-448, doi:10.1146/annurev-pathol-012414-040424 (2015). [0291] 49 Dobbelstein, M. & Sorensen, C. S. Exploiting replicative stress to treat cancer. Nature Reviews Drug Discovery 14, 405-423, doi:10.1038/nrd4553 (2015). [0292] 50 Nieto-Soler, M. et al. Efficacy of ATR inhibitors as single agents in Ewing sarcoma. Oncotarget 7, 58759-58767, doi:10.18632/oncotarget.11643 (2016). [0293] 51 Bartkova, J. et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864-870, doi:10.1038/nature03482 (2005). [0294] 52 Bartek, J., Mistrik, M. & Bartkova, J. Thresholds of replication stress signaling in cancer development and treatment. Nat Struct Mol Biol 19, 5-7, doi:10.1038/nsmb.2220 (2012). [0295] 53 Smith, Z. D. et al. Epigenetic restriction of extraembryonic lineages mirrors the somatic transition to cancer. Nature 549, 543-547, doi:10.1038/nature23891 (2017).