Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer

•Aberrant acetylated regions (AARs) are an epigenetic barrier in SCNT embryos•TSA partly fixes AARs but is restricted by native epi-environments donor cells•Dux restores H3K9ac occupancy, drives 2C activation, and improves efficiency•Rescue efficiency Kdm4b TSA largely relies on Dux cluster activation Differentiated somatic cells can be reprogrammed to totipotent embryos through cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found improve efficiency, the underlying mechanism remains undetermined. Here, we examined genome-wide during embryo development that aberrant resulted reduced 2-cell genome activation. treatment corrects acetylation dictated environment. We further identified overexpression of greatly correcting signal at its target sites, ensuring appropriate Intriguingly, improvement mediated impeded knockout embryos. Together, our study reveals reprogramming important for optimal identifies as a crucial transcription factor this process. oocyte cytoplasm capable regain totipotency (SCNT), which enables animal cloning generation embryonic stem (ntESCs) (Chen et al., 2020Chen M. Zhu Q. Li C. Kou X. Zhao Y. Xu R. Yang L. Gu al.Chromatin architecture reorganization murine embryos.Nat. Commun. 2020; 11: 1813Crossref PubMed Scopus (15) Google Scholar; Chung 2014Chung Y.G. Eum J.H. Lee J.E. Shim S.H. Sepilian V. Hong S.W. Treff N.R. Choi Y.H. Kimbrel E.A. al.Human using adult cells.Cell Stem Cell. 2014; 14: 777-780Abstract Full Text PDF (129) Liu 2018Liu Z. Cai Wang Nie Zhang Lu Poo Sun Cloning macaque monkeys transfer.Cell. 2018; 172: 881-887.e7Abstract (122) Tachibana 2013Tachibana Amato P. Sparman Gutierrez N.M. Tippner-Hedges Ma H. Kang E. Fulati A. H.-S. Sritanaudomchai derived 2013; 153: 1228-1238Abstract (517) Wakayama 1998Wakayama T. Perry A.C.F. Zuccotti Johnson K.R. Yanagimachi Full-term mice from enucleated oocytes injected cumulus nuclei.Nature. 1998; 394: 369-374Crossref (1895) Wilmut 1997Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H.S. Viable offspring fetal mammalian cells.Nature. 1997; 385: 810-813Crossref (3903) Scholar). Thus, holds great potential producing gene-edited models biomedical research conserving endangered animals (Chung Scholar, 2015Chung Matoba S. F. Jiang W. Cha K.Y. D.R. Histone demethylase expression enhances human promotes derivation pluripotent 2015; 17: 758-766Abstract (102) Munsie 2000Munsie M.J. Michalska O’Brien C.M. Trounson A.O. Pera M.F. Mountford P.S. Isolation mouse nuclei.Curr. Biol. 2000; 10: 989-992Abstract (302) Niu 2017Niu D. Wei H.-J. Lin George I.H. H.-Y. Kan Shrock al.Inactivation porcine endogenous retrovirus pigs CRISPR-Cas9.Science. 2017; 357: 1303-1307Crossref (316) Qiu 2019Qiu Huang Cheng al.BMAL1 display sleep psychiatric disorders.Natl. Sci. Rev. 2019; 6: 87-100Crossref (38) Yan 2018Yan Tu Fan N. Ouyang Lai al.A Huntingtin knockin pig model recapitulates features selective neurodegeneration Huntington’s disease.Cell. 173: 989-1002.e13Abstract (111) 2001Wakayama Tabar Rodriguez Studer Mombaerts Differentiation lines generated transfer.Science. 2001; 292: 740-743Crossref (484) Yu 2018Yu Long K. Guo Qing Pan Jia B. al.Generation GHR-modified Laron syndrome via dual-sgRNAs/Cas9 system transfer.J. Transl. Med. 16: 41Crossref (4) However, extremely terms blastocyst formation full-term (Yang 2007Yang Smith S.L. Tian X.C. Lewin H.A. Renard J.-P. Nuclear cloned implications therapeutic cloning.Nat. Genet. 2007; 39: 295-302Crossref (456) Recently, researchers have developed multiple methods removing barriers. Injection mRNAs Kdm4d or Kdm4b, both H3K9me3 demethylases, into term less than 1% more 8% (Liu 2016aLiu Gao Wu Xiu al.Identification key factors conquering developmental arrest combining biopsy single-cell sequencing.Cell Discov. 2016; 2: 16010Crossref (104) 2014Matoba Iwabuchi K.A. Shen Inoue Embryonic following persisting methylation.Cell. 159: 884-895Abstract (244) use (HDACi), (TSA), increase overall 6% (Kishigami 2006Kishigami Mizutani Ohta Hikichi Thuan N.V. Bui H.T. Significant technique after transfer.Biochem. Biophys. Res. 2006; 340: 183-189Crossref (461) Rybouchkin 2006Rybouchkin Kato Tsunoda Role nuclei transfer.Biol. Reprod. 74: 1083-1089Crossref (239) it unexplored how HDACi reprogramming. results previous showed combination injection could not suggesting function might overlap removal (Matoba Although interventions adjust modifications significantly overcome (2C) allow comparable naturally fertilized (NF) embryos, still low, indicating abnormal implantation. Indeed, extraembryonic tissues, such placenta larger those NF Xist aberrantly activated, leading X chromosome inactivation (Inoue 2010Inoue Kohda Sugimoto Sado Ogonuki Shiura Ikeda Mochida Fujii al.Impeding active 2010; 330: 496-499Crossref (183) Recent studies revealed (TF) double homeobox (Dux DUX4 human) positive TF activating chromatin landscape regulating sets 2C-specific genes (De Iaco 2017De Planet Coluccio Verp Duc Trono DUX-family regulate zygotic placental mammals.Nat. 49: 941-945Crossref (205) Hendrickson 2017Hendrickson P.G. Doráis J.A. Grow E.J. Whiddon J.L. Lim J.-W. Wike C.L. Weaver B.D. Pflueger Emery B.R. Wilcox A.L. al.Conserved roles DUX cleavage-stage MERVL/HERVL retrotransposons.Nat. 925-934Crossref (235) 2017Whiddon Langford A.T. Wong C.-J. Zhong J.W. Tapscott S.J. Conservation innovation DUX4-family gene network.Nat. 935-940Crossref In addition, recent Dux-miR-344-Zmym2 regulatory pathway governing 2020aYang Zang Chen Fidalgo Sanchez-Priego Caichen Macfarlan al.DUX-miR-344-ZMYM2-mediated MERVL LTRs induces 2C-like state.Cell 26: 234-250.e7Abstract (21) high similarity within two DNA-binding homeodomains C-terminal domain. recruit acetyltransferases EP300 CBP (CREB binding protein) domain induce local relaxation transcriptional nearby genes. Ectopic was proven cause global hyperacetylation H3. Both transiently expressed before onset (ZGA) (Choi 2016Choi Gearhart M.D. Cui Bosnakovski Kim Schennum Kyba recruits p300/CBP C-terminus H3K27 changes.Nucleic Acids 44: 5161-5173Crossref (71) Ziegenhain 2017Ziegenhain Vieth Parekh Reinius Guillaumet-Adkins Smets Leonhardt Heyn Hellmann Enard Comparative analysis RNA sequencing methods.Mol. 65: 631-643.e4Abstract (551) precise temporal regulation development, demonstrated prolonged blastomeres signatures. It intriguing maternal (KO) (MZ-KO) survive adulthood, complicated role embryogenesis Zhang, 2019Chen Loss causes minor defects compatible development.Nat. 51: 947-951Crossref (52) 2019Guo Zhou Bi al.Precise development.Cell 29: 956-959Crossref (24) early elusive. Still, utilized potent tool generating vitro. Epigenetic plays critical Aberrant H3K9me3, H3K4me3, DNA re-methylation were barriers limited (Gao 2018Gao Liao Bai Qiao al.Inhibition post-implantation embryos.Cell 23: 426-435.e5Abstract (39) Besides, loss H3K27me3 imprinting And monoallelic deletion H3K27me3-imprinted 2020Inoue Kamimura Hirose Honda Miura Hada Hasegawa al.Loss Sfmbt2 miRNA enlargement placentas.Nat. 2150Crossref (14) 2018Matoba Press J.T. disrupts 343-354.e5Abstract (53) 2020Wang L.-Y. Z.-K. L.-B. X.-H. Feng G.-H. J.-Q. Y.-F. al.Overcoming intrinsic transfer.Cell 27: 315-325.e5Abstract (12) Meanwhile, lysine core histones undergoes dramatic changes SCNT. As indicated work, intensity much lower normal elevated level intracytoplasmic sperm (ICSI) turn contributed improved (Wang 2007Wang Dynamic methylation first cycle embryos.Biol. 77: 1007-1016Crossref (141) Additionally, marks state promoters directly boosts elongation polymerase II (Pol II) (Gates 2017Gates L.A. Shi Rohira A.D. Bedford M.T. Sagum C.A. Jung S.Y. Qin Tsai M.-J. al.Acetylation H3 9 mediates switch initiation elongation.J. Chem. 14456-14472Abstract (84) affects TFs consequently worsens enigmatic. study, high-resolution map vitro fertilization (IVF)/NF 1-cell morula stage ultra-low-input immunoprecipitation followed (ULI-NChIP-seq) explored difference occupancy between IVF/NF barrier, contain many downregulated silenced fix most effects restrained environments cells. significant enrichment motif hyper-H3K9ac signals specifically (e2C) IVF e2C Strikingly, full-length mRNA AARs, vivo. Moreover, essential promote also dependent Dux. Our accurate modulation provides novel approach mice, ?70% arrested cleavage stages Ogura 2013Ogura advancements transfer.Philos. Trans. Soc. Lond. B 368: 20110329Crossref (131) defective modification compared ICSI whereas efficiently defect, especially 1C To address this, used ULI-NChIP-seq generate (Figure 1A). profiles highly correlated ENCODE project massive S1A). few 200 fully recapitulate peaks S1B). reproducible replicates S1C). accordance observed hypo-H3K9ac quickly increased H3K9ac. removal, declined (Figures S1D–S1G). then scanned 5-kb sliding window identify each stage. termed (AHARs). Similarly, (ALARs). AHARs ALARs together make up (refer Data S1). number precipitously decreased 1B 1C). Consistently, covered genomic cycles, line immunostaining S1D–S1G S2A). Of note, accounted small fraction late (l2C) These findings imply these successfully l2C long develop later stages. Alternatively, fail reported enriched reprogramming-resistant (RRRs) Consistent that, RRRs S2B). DNase I depleted 1D; Djekidel 2018Djekidel M.N. Suzuki Reprogramming accessibility replication independent.Cell Rep. 1939-1947Abstract (17) This indicates states impact Further inherited resisted contrast, only ?37% S2C). suggested majority faithfully remodeled new formed well fate determination Particularly, center (XIC) became higher 1E S2D). levels chromosomes similar clearly loci artifact dose (XX cells) (both XX XY). abnormalities entering differentiation, account rate influence investigate calculated different throughout S2E). Interestingly, pattern opposite S2F S2G). finding suggests impediment TSA, HDAC inhibitor, widely (Akagi 2011Akagi Matsukawa Fukunari Kaneda Watanabe Takahashi Treatment preimplantation bovine embryos.J. Dev. 2011; 57: 120-126Crossref (47) Kishigami 2010Zhao Hao Ross Spate L.D. Walters E.M. Samuel M.S. Rieke Murphy C.N. Prather R.S. inhibitors vivo competence embryos.Cell. Reprogram. 12: 75-83Crossref (120) order understand profiled upon treatment. There hundred differentially treated without S3A). consistent showing 2015Inoue Oikawa Nakamura Nakano Abe Trichostatin produced transfer.Sci. 5: 10127Crossref Furthermore, heterogeneity recovery. For example, rescued some genes, others, Sall4 Ezh2, exhibited partial rescue. certain failed rescued, Zscan4d S3B; Eckersley-Maslin 2016Eckersley-Maslin M.A. Svensson Krueger Stubbs T.M. Giehr Miragaia R.J. Kyriakopoulos Berrens R.V. Milagre al.MERVL/Zscan4 network transient demethylation mESCs.Cell 179-192Abstract (87) Falco 2007Falco G. S.-L. Stanghellini Bassey U.C. Hamatani Ko M.S.H. Zscan4: exclusively cells.Dev. 307: 539-550Crossref (155) Zalzman 2010Zalzman Sharova L.V. Nishiyama Thomas Stagg Hoang H.G. H.-T. Indig F.E. al.Zscan4 regulates telomere stability ES 464: 858-863Crossref (251) extensive S1D–S1G, S2A, S2B; Table Therefore, next altered Surprisingly, restored resembling S3C). there existed fixed