Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells

Abstract

The use of two inhibitors of Mek1/2 and Gsk3β (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that the use of a two-step a2i (alternative two inhibitors of Src and Gsk3β, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2-itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can longterm maintain paternal imprints.

Keywords

paternal imprints / androgenetic haploid ESCs / DMRs / semi-cloned mice / alternative 2i

References

[1] AtlasiY, Stunnenberg HG (2017) The interplay of epigenetic marks during stem cell differentiation and development. Nat Rev Genet 18:643–658

[2] BarlowDP, Bartolomei MS (2014) Genomic imprinting in mammals. Cold Spring Harb Perspect Biol 6:a018382

[3] ChoiJ, Clement K, HuebnerAJ, WebsterJ, RoseCM, BrumbaughJ, Walsh RM, LeeS, SavolA, Etchegaray JP et al (2017a) DUSP9 modulates DNA hypomethylation in female mouse pluripotent stem cells. Cell Stem Cell 20:706–719

[4] ChoiJ, Huebner AJ, ClementK, WalshRM, SavolA, LinK, GuH, Di StefanoB, Brumbaugh J, KimSY et al (2017b) Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells. Nature 548:219–223

[5] DeanW, BowdenL, AitchisonA, Klose J, MooreT, MenesesJJ, ReikW, FeilR (1998) Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125:2273–2282

[6] DuJ, Johnson LM, JacobsenSE, PatelDJ (2015) DNA methylation pathways and their crosstalk with histone methylation. Nat Rev Mol Cell Biol 16:519–532

[7] EdwardsCA, Ferguson-Smith AC (2007) Mechanisms regulating imprinted genes in clusters. Curr Opin Cell Biol 19:281–289

[8] EllingU, Taubenschmid J, WirnsbergerG, O’MalleyR, Demers SP, VanhaelenQ, ShukalyukAI, Schmauss G, SchramekD, SchnuetgenF et al (2011) Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell Stem Cell 9:563–574

[9] EvansMJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156

[10] Ferguson-SmithAC (2011) Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet 12:565–575

[11] GuoX, WangL, LiJ, DingZ, XiaoJ, Yin X, HeS, ShiP, DongL, LiG et al (2015) Structural insight into autoinhibition and histone H3-induced activation of DNMT3A. Nature 517:640–644

[12] HabibiE, Brinkman AB, ArandJ, KroezeLI, Kerstens HH, MatareseF, LepikhovK, GutM, Brun-HeathI, Hubner NC et al (2013) Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 13:360–369

[13] HernandezL, KozlovS, PirasG, Stewart CL (2003) Paternal and maternal genomes confer opposite effects on proliferation, cellcycle length, senescence, and tumor formation. Proc Natl Acad Sci USA 100:13344–13349

[14] HumpherysD, EgganK, AkutsuH, Hochedlinger K, RideoutWM 3rd, BiniszkiewiczD, Yanagimachi R, JaenischR (2001) Epigenetic instability in ES cells and cloned mice. Science 293:95–97

[15] KeniryA, OxleyD, MonnierP, Kyba M, DandoloL, SmitsG, ReikW (2012) The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r. Nat Cell Biol 14:659–665

[16] KiyonariH, KanekoM, AbeS, AizawaS (2010) Three inhibitors of FGF receptor, ERK, and GSK3 establishes germline-competent embryonic stem cells of C57BL/6N mouse strain with high efficiency and stability. Genesis 48:317–327

[17] LeeJ, Matsuzawa A, ShiuraH, SutaniA, IshinoF (2018) Preferable in vitro condition for maintaining faithful DNA methylation imprinting in mouse embryonic stem cells. Genes Cells 23:146–160

[18] LeebM, WutzA (2011) Derivation of haploid embryonic stem cells from mouse embryos. Nature 479:131–134

[19] LeebM, Dietmann S, ParamorM, NiwaH, SmithA (2014) Genetic exploration of the exit from self-renewal using haploid embryonic stem cells. Cell Stem Cell 14:385–393

[20] LiY, LiJ (2019) Technical advances contribute to the study of genomic imprinting. PLoS Genet 15:e1008151

[21] LiX, ItoM, ZhouF, Youngson N, ZuoX, LederP, Ferguson-Smith AC (2008) A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints. Dev Cell 15:547–557

[22] LiW, ShuaiL, WanH, DongM, WangM, Sang L, FengC, LuoGZ, LiT, LiX et al (2012) Androgenetic haploid embryonic stem cells produce live transgenic mice. Nature 490:407–411

[23] LiQ, LiY, YinQ, HuangS, WangK, Zhuo L, LiW, ChangB, LiJ (2020) Temporal regulation of prenatal embryonic development by paternal imprinted loci. Sci China Life Sci 63:1–17

[24] MartelloG, SmithA (2014) The nature of embryonic stem cells. Annu Rev Cell Dev Biol 30:647–675

[25] NagyA, GoczaE, DiazEM, Prideaux VR, IvanyiE, MarkkulaM, Rossant J (1990) Embryonic stem cells alone are able to support fetal development in the mouse. Development 110:815–821

[26] NagyA, Rossant J, NagyR, Abramow-NewerlyW, Roder JC (1993) Derivation of completely cell culture-derived mice from earlypassage embryonic stem cells. Proc Natl Acad Sci USA 90:8424–8428

[27] NicholsJ, JonesK, PhillipsJM, Newland SA, RoodeM, MansfieldW, SmithA, CookeA (2009) Validated germline-competent embryonic stem cell lines from nonobese diabetic mice. Nat Med 15:814–818

[28] OoiSK, QiuC, BernsteinE, Li K, JiaD, YangZ, Erdjument-Bromage H, TempstP, LinSP, AllisCD et al (2007) DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 448:714–717

[29] QuennevilleS, VerdeG, CorsinottiA, Kapopoulou A, JakobssonJ, OffnerS, Baglivo I, PedonePV, GrimaldiG, RiccioA et al (2011) In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions. Mol Cell 44:361–372

[30] SagiI, ChiaG, Golan-LevT, Peretz M, WeissbeinU, SuiL, SauerMV, YanukaO, Egli D, BenvenistyN (2016) Derivation and differentiation of haploid human embryonic stem cells. Nature 532:107–111

[31] ShiH, Strogantsev R, TakahashiN, KazachenkaA, Lorincz MC, HembergerM, Ferguson-SmithAC (2019) ZFP57 regulation of transposable elements and gene expression within and beyond imprinted domains. Epigenetics Chromatin 12:49

[32] ShimizuT, UedaJ, HoJC, Iwasaki K, PoellingerL, HaradaI, SawadaY (2012) Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling. Stem Cells 30:1394–1404

[33] StrogantsevR, Krueger F, YamazawaK, ShiH, GouldP, Goldman-RobertsM, McEwenK, SunB, PedersenR, Ferguson-Smith AC (2015) Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression. Genome Biol 16:112

[34] TakahashiN, GrayD, StrogantsevR, NoonA, Delahaye C, SkarnesWC, TatePH, Ferguson-Smith AC (2015) ZFP57 and the targeted maintenance of postfertilization genomic imprints. Cold Spring Harb Symp Quant Biol 80:177–187

[35] TuckerKL, BeardC, DausmannJ, Jackson-Grusby L, LairdPW, LeiH, LiE, JaenischR (1996) Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev 10:1008–1020

[36] WalterM, Teissandier A, Perez-PalaciosR, Bourc’hisD (2016) An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells. Elife 5:e11418

[37] WangZQ, KieferF, UrbanekP, Wagner EF (1997) Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection. Mech Dev 62:137–145

[38] WangC, LiuX, GaoY, YangL, LiC, LiuW, ChenC, Kou X, ZhaoY, ChenJ et al (2018) Reprogramming of H3K9me3-dependent heterochromatin during mammalian embryo development. Nat Cell Biol 20:620–631

[39] WangQ, YuG, MingX, Xia W, XuX, ZhangY, ZhangW, LiY, HuangC, XieH et al (2020) Imprecise DNMT1 activity coupled with neighbor-guided correction enables robust yet flexible epigenetic inheritance. Nat Genet 52:828–839

[40] WangYH, YanM, ZhangX, Liu XY, DingYF, LaiCP, TongMH, LiJS (2021) Rescue of male infertility through correcting a genetic mutation causing meiotic arrest in spermatogonial stem cells. Asian J Androl

[41] WuY, LiangD, WangY, Bai M, TangW, BaoS, YanZ, LiD, LiJ (2013) Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13:659–662

[42] WuB, LiL, LiB, GaoJ, ChenY, Wei M, YangZ, ZhangB, LiS, LiK et al (2020) Activin A and BMP4 signaling expands potency of mouse embryonic stem cells in serum-free media. Stem Cell Rep 14:241–255

[43] WuB, LiY, LiB, ZhangB, WangY, Li L, GaoJ, FuY, LiS, ChenC et al (2021) DNMTs play an important role in maintaining the pluripotency of leukemia inhibitory factor-dependent embryonic stem cells. Stem Cell Rep 16:582–596

[44] YagiM, Kishigami S, TanakaA, SemiK, Mizutani E, WakayamaS, WakayamaT, Yamamoto T, YamadaY (2017) Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation. Nature 548:224–227

[45] YangH, ShiL, WangBA, Liang D, ZhongC, LiuW, NieY, LiuJ, ZhaoJ, GaoX et al (2012) Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149:605–617

[46] YangH, LiuZ, MaY, ZhongC, YinQ, ZhouC, ShiL, CaiY, ZhaoH, Wang H et al (2013) Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes. Cell Res 23:1187–1200

[47] YingQL, WrayJ, NicholsJ, Batlle-Morera L, DobleB, WoodgettJ, CohenP, SmithA (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523

[48] ZhangM, LiuY, LiuG, LiX, JiaY, SunL, WangL, Zhou Q, HuangY (2015) Rapidly generating knockoutmice fromH19-Igf2 engineered androgenetic haploid embryonic stem cells. Cell Discov 1:15031

[49] ZhangXM, WuK, ZhengY, Zhao H, GaoJ, HouZ, ZhangM, LiaoJ, Zhang J, GaoY et al (2020) In vitro expansion of human sperm through nuclear transfer. Cell Res 30:356–359

[50] ZhongC, YinQ, XieZ, BaiM, DongR, Tang W, XingYH, ZhangH, YangS, ChenLL et al (2015) CRISPR-Cas9-mediated genetic screening in mice with haploid embryonic stem cells carrying a guide RNA library. Cell Stem Cell 17:221–232

[51] ZhongC, XieZ, YinQ, DongR, YangS, Wu Y, YangL, LiJ (2016a) Parthenogenetic haploid embryonic stem cells efficiently support mouse generation by oocyte injection. Cell Res 26:131–134

[52] ZhongC, ZhangM, YinQ, ZhaoH, WangY, Huang S, TaoW, WuK, ChenZJ, LiJ (2016b) Generation of human haploid embryonic stem cells from parthenogenetic embryos obtained by microsurgical removal of male pronucleus. Cell Res 26:743–746

[53] ZwakaTP (2017) Stem cells: the cost of perpetual youth. Nature 548:165–166

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