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• W3092364740 abstract "•Aberrant acetylated regions (AARs) are an epigenetic barrier in SCNT embryos•TSA partly fixes AARs but is restricted by native epi-environments in donor cells•Dux restores H3K9ac occupancy, drives 2C activation, and improves SCNT efficiency•Rescue efficiency by Kdm4b and TSA largely relies on Dux cluster activation Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process. Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process. The oocyte cytoplasm is capable of reprogramming somatic cells to regain totipotency through somatic cell nuclear transfer (SCNT), which enables animal cloning and generation of nuclear transfer embryonic stem cells (ntESCs) (Chen et al., 2020Chen M. Zhu Q. Li C. Kou X. Zhao Y. Li Y. Xu R. Yang L. Yang L. Gu L. et al.Chromatin architecture reorganization in murine somatic cell nuclear transfer embryos.Nat. Commun. 2020; 11: 1813Crossref PubMed Scopus (15) Google Scholar; Chung et al., 2014Chung Y.G. Eum J.H. Lee J.E. Shim S.H. Sepilian V. Hong S.W. Lee Y. Treff N.R. Choi Y.H. Kimbrel E.A. et al.Human somatic cell nuclear transfer using adult cells.Cell Stem Cell. 2014; 14: 777-780Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar; Liu et al., 2018Liu Z. Cai Y. Wang Y. Nie Y. Zhang C. Xu Y. Zhang X. Lu Y. Wang Z. Poo M. Sun Q. Cloning of macaque monkeys by somatic cell nuclear transfer.Cell. 2018; 172: 881-887.e7Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Gutierrez N.M. Tippner-Hedges R. Ma H. Kang E. Fulati A. Lee H.-S. Sritanaudomchai H. et al.Human embryonic stem cells derived by somatic cell nuclear transfer.Cell. 2013; 153: 1228-1238Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar; Wakayama et al., 1998Wakayama T. Perry A.C.F. Zuccotti M. Johnson K.R. Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.Nature. 1998; 394: 369-374Crossref PubMed Scopus (1895) Google Scholar; Wilmut et al., 1997Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H.S. Viable offspring derived from fetal and adult mammalian cells.Nature. 1997; 385: 810-813Crossref PubMed Scopus (3903) Google Scholar). Thus, SCNT holds great potential for producing gene-edited animal models for biomedical research and conserving endangered animals (Chung et al., 2014Chung Y.G. Eum J.H. Lee J.E. Shim S.H. Sepilian V. Hong S.W. Lee Y. Treff N.R. Choi Y.H. Kimbrel E.A. et al.Human somatic cell nuclear transfer using adult cells.Cell Stem Cell. 2014; 14: 777-780Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, Chung et al., 2015Chung Y.G. Matoba S. Liu Y. Eum J.H. Lu F. Jiang W. Lee J.E. Sepilian V. Cha K.Y. Lee D.R. Zhang Y. Histone demethylase expression enhances human somatic cell nuclear transfer efficiency and promotes derivation of pluripotent stem cells.Cell Stem Cell. 2015; 17: 758-766Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar; Liu et al., 2018Liu Z. Cai Y. Wang Y. Nie Y. Zhang C. Xu Y. Zhang X. Lu Y. Wang Z. Poo M. Sun Q. Cloning of macaque monkeys by somatic cell nuclear transfer.Cell. 2018; 172: 881-887.e7Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; Munsie et al., 2000Munsie M.J. Michalska A.E. O’Brien C.M. Trounson A.O. Pera M.F. Mountford P.S. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.Curr. Biol. 2000; 10: 989-992Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar; Niu et al., 2017Niu D. Wei H.-J. Lin L. George H. Wang T. Lee I.H. Zhao H.-Y. Wang Y. Kan Y. Shrock E. et al.Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9.Science. 2017; 357: 1303-1307Crossref PubMed Scopus (316) Google Scholar; Qiu et al., 2019Qiu P. Jiang J. Liu Z. Cai Y. Huang T. Wang Y. Liu Q. Nie Y. Liu F. Cheng J. et al.BMAL1 knockout macaque monkeys display reduced sleep and psychiatric disorders.Natl. Sci. Rev. 2019; 6: 87-100Crossref Scopus (38) Google Scholar; Yan et al., 2018Yan S. Tu Z. Liu Z. Fan N. Yang H. Yang S. Yang W. Zhao Y. Ouyang Z. Lai C. et al.A Huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington’s disease.Cell. 2018; 173: 989-1002.e13Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar; Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Gutierrez N.M. Tippner-Hedges R. Ma H. Kang E. Fulati A. Lee H.-S. Sritanaudomchai H. et al.Human embryonic stem cells derived by somatic cell nuclear transfer.Cell. 2013; 153: 1228-1238Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar; Wakayama et al., 2001Wakayama T. Tabar V. Rodriguez I. Perry A.C.F. Studer L. Mombaerts P. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer.Science. 2001; 292: 740-743Crossref PubMed Scopus (484) Google Scholar; Yu et al., 2018Yu H. Long W. Zhang X. Xu K. Guo J. Zhao H. Li H. Qing Y. Pan W. Jia B. et al.Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer.J. Transl. Med. 2018; 16: 41Crossref PubMed Scopus (4) Google Scholar). However, the cloning efficiency is extremely low in terms of blastocyst formation and full-term development (Yang et al., 2007Yang X. Smith S.L. Tian X.C. Lewin H.A. Renard J.-P. Wakayama T. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning.Nat. Genet. 2007; 39: 295-302Crossref PubMed Scopus (456) Google Scholar). Recently, researchers have developed multiple methods to improve SCNT efficiency by removing epigenetic barriers. Injection of the mRNAs of Kdm4d or Kdm4b, both H3K9me3 demethylases, into cloned mouse embryos can improve the term development from less than 1% to more than 8% (Liu et al., 2016aLiu W. Liu X. Wang C. Gao Y. Gao R. Kou X. Zhao Y. Li J. Wu Y. Xiu W. et al.Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing.Cell Discov. 2016; 2: 16010Crossref PubMed Scopus (104) Google Scholar; Matoba et al., 2014Matoba S. Liu Y. Lu F. Iwabuchi K.A. Shen L. Inoue A. Zhang Y. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.Cell. 2014; 159: 884-895Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The use of a histone deacetylase inhibitor (HDACi), trichostatin A (TSA), can increase the overall cloning efficiency from 1% to 6% (Kishigami et al., 2006Kishigami S. Mizutani E. Ohta H. Hikichi T. Thuan N.V. Wakayama S. Bui H.T. Wakayama T. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.Biochem. Biophys. Res. Commun. 2006; 340: 183-189Crossref PubMed Scopus (461) Google Scholar; Rybouchkin et al., 2006Rybouchkin A. Kato Y. Tsunoda Y. Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer.Biol. Reprod. 2006; 74: 1083-1089Crossref PubMed Scopus (239) Google Scholar). However, it remains unexplored how HDACi treatment improves SCNT reprogramming. The results from a previous study showed that the combination of Kdm4d injection and TSA treatment could not further increase the cloning efficiency, suggesting that the function of TSA treatment might overlap with H3K9me3 removal (Matoba et al., 2014Matoba S. Liu Y. Lu F. Iwabuchi K.A. Shen L. Inoue A. Zhang Y. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.Cell. 2014; 159: 884-895Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Although interventions to adjust epigenetic modifications can significantly overcome 2-cell (2C) arrest and allow blastocyst development of SCNT embryos comparable to that of naturally fertilized (NF) embryos, the overall cloning efficiency is still low, indicating that the SCNT embryos are abnormal after implantation. Indeed, extraembryonic tissues, such as the placenta of SCNT embryos, are larger than those of NF embryos, and Xist could be aberrantly activated, leading to aberrant X chromosome inactivation (Inoue et al., 2010Inoue K. Kohda T. Sugimoto M. Sado T. Ogonuki N. Matoba S. Shiura H. Ikeda R. Mochida K. Fujii T. et al.Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.Science. 2010; 330: 496-499Crossref PubMed Scopus (183) Google Scholar). Recent studies have revealed the transcription factor (TF) double homeobox (Dux in mouse and DUX4 in human) as a positive TF in activating the mammalian embryonic genome chromatin landscape and regulating the expression of sets of 2C-specific genes (De Iaco et al., 2017De Iaco A. Planet E. Coluccio A. Verp S. Duc J. Trono D. DUX-family transcription factors regulate zygotic genome activation in placental mammals.Nat. Genet. 2017; 49: 941-945Crossref PubMed Scopus (205) Google Scholar; Hendrickson et al., 2017Hendrickson P.G. Doráis J.A. Grow E.J. Whiddon J.L. Lim J.-W. Wike C.L. Weaver B.D. Pflueger C. Emery B.R. Wilcox A.L. et al.Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.Nat. Genet. 2017; 49: 925-934Crossref PubMed Scopus (235) Google Scholar; Whiddon et al., 2017Whiddon J.L. Langford A.T. Wong C.-J. Zhong J.W. Tapscott S.J. Conservation and innovation in the DUX4-family gene network.Nat. Genet. 2017; 49: 935-940Crossref PubMed Scopus (122) Google Scholar). In addition, our recent study identified a Dux-miR-344-Zmym2 regulatory pathway governing totipotency (Yang et al., 2020aYang F. Huang X. Zang R. Chen J. Fidalgo M. Sanchez-Priego C. Yang J. Caichen A. Ma F. Macfarlan T. et al.DUX-miR-344-ZMYM2-mediated activation of MERVL LTRs induces a totipotent 2C-like state.Cell Stem Cell. 2020; 26: 234-250.e7Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). DUX4 and Dux have high similarity within the two DNA-binding homeodomains and the C-terminal domain. DUX4 can recruit the histone acetyltransferases EP300 and CBP (CREB binding protein) by its C-terminal domain and induce local chromatin relaxation and transcriptional activation of nearby genes. Ectopic expression of both DUX4 and Dux was proven to cause global hyperacetylation of histone H3. Both are transiently expressed before the global onset of zygotic genome activation (ZGA) (Choi et al., 2016Choi S.H. Gearhart M.D. Cui Z. Bosnakovski D. Kim M. Schennum N. Kyba M. DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes.Nucleic Acids Res. 2016; 44: 5161-5173Crossref PubMed Scopus (71) Google Scholar; Hendrickson et al., 2017Hendrickson P.G. Doráis J.A. Grow E.J. Whiddon J.L. Lim J.-W. Wike C.L. Weaver B.D. Pflueger C. Emery B.R. Wilcox A.L. et al.Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.Nat. Genet. 2017; 49: 925-934Crossref PubMed Scopus (235) Google Scholar; Ziegenhain et al., 2017Ziegenhain C. Vieth B. Parekh S. Reinius B. Guillaumet-Adkins A. Smets M. Leonhardt H. Heyn H. Hellmann I. Enard W. Comparative analysis of single-cell RNA sequencing methods.Mol. Cell. 2017; 65: 631-643.e4Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). The precise temporal regulation of Dux is crucial for embryonic development, and we have demonstrated that prolonged overexpression of Dux can induce developmental arrest of blastomeres with 2C signatures. It is intriguing that Dux maternal and zygotic knockout (KO) (MZ-KO) embryos can survive to adulthood, indicating its more complicated role during embryogenesis (Chen and Zhang, 2019Chen Z. Zhang Y. Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development.Nat. Genet. 2019; 51: 947-951Crossref PubMed Scopus (52) Google Scholar; Guo et al., 2019Guo M. Zhang Y. Zhou J. Bi Y. Xu J. Xu C. Kou X. Zhao Y. Li Y. Tu Z. et al.Precise temporal regulation of Dux is important for embryo development.Cell Res. 2019; 29: 956-959Crossref PubMed Scopus (24) Google Scholar). The function of Dux in early embryo development remains elusive. Still, Dux has been utilized as a potent tool for generating 2C-like cells in vitro. Epigenetic reprogramming plays a critical role in the development of SCNT embryos. Aberrant H3K9me3, H3K4me3, and DNA re-methylation were proven to be potent barriers that greatly limited full-term development of SCNT embryos (Gao et al., 2018Gao R. Wang C. Gao Y. Xiu W. Chen J. Kou X. Zhao Y. Liao Y. Bai D. Qiao Z. et al.Inhibition of aberrant DNA re-methylation improves post-implantation development of somatic cell nuclear transfer embryos.Cell Stem Cell. 2018; 23: 426-435.e5Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar; Liu et al., 2016aLiu W. Liu X. Wang C. Gao Y. Gao R. Kou X. Zhao Y. Li J. Wu Y. Xiu W. et al.Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing.Cell Discov. 2016; 2: 16010Crossref PubMed Scopus (104) Google Scholar; Matoba et al., 2014Matoba S. Liu Y. Lu F. Iwabuchi K.A. Shen L. Inoue A. Zhang Y. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.Cell. 2014; 159: 884-895Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Besides, loss of H3K27me3 imprinting in somatic cells impeded the development of SCNT embryos. And monoallelic deletion of the key H3K27me3-imprinted genes could largely improve SCNT efficiency (Inoue et al., 2020Inoue K. Ogonuki N. Kamimura S. Inoue H. Matoba S. Hirose M. Honda A. Miura K. Hada M. Hasegawa A. et al.Loss of H3K27me3 imprinting in the Sfmbt2 miRNA cluster causes enlargement of cloned mouse placentas.Nat. Commun. 2020; 11: 2150Crossref PubMed Scopus (14) Google Scholar; Matoba et al., 2018Matoba S. Wang H. Jiang L. Lu F. Iwabuchi K.A. Wu X. Inoue K. Yang L. Press W. Lee J.T. et al.Loss of H3K27me3 imprinting in somatic cell nuclear transfer embryos disrupts post-implantation development.Cell Stem Cell. 2018; 23: 343-354.e5Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; Wang et al., 2020Wang L.-Y. Li Z.-K. Wang L.-B. Liu C. Sun X.-H. Feng G.-H. Wang J.-Q. Li Y.-F. Qiao L.-Y. Nie H. et al.Overcoming intrinsic H3K27me3 imprinting barriers improves post-implantation development after somatic cell nuclear transfer.Cell Stem Cell. 2020; 27: 315-325.e5Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Meanwhile, the lysine acetylation on core histones undergoes dramatic changes following SCNT. As indicated in our previous work, the H3K9ac intensity in SCNT embryos was much lower than that in the normal embryos. TSA treatment elevated the H3K9ac signal of SCNT embryos to a level comparable to that of intracytoplasmic sperm injection (ICSI) embryos, which in turn contributed to the improved development of SCNT embryos (Wang et al., 2007Wang F. Kou Z. Zhang Y. Gao S. Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos.Biol. Reprod. 2007; 77: 1007-1016Crossref PubMed Scopus (141) Google Scholar). Additionally, H3K9ac marks the active state of promoters and directly boosts the transcriptional elongation of RNA polymerase II (Pol II) (Gates et al., 2017Gates L.A. Shi J. Rohira A.D. Feng Q. Zhu B. Bedford M.T. Sagum C.A. Jung S.Y. Qin J. Tsai M.-J. et al.Acetylation on histone H3 lysine 9 mediates a switch from transcription initiation to elongation.J. Biol. Chem. 2017; 292: 14456-14472Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). However, how aberrant acetylation affects the function of key TFs and consequently worsens SCNT efficiency remains enigmatic. In this study, we generated a high-resolution H3K9ac map of both in vitro fertilization (IVF)/NF and SCNT embryos from the 1-cell embryo to the morula stage using ultra-low-input chromatin immunoprecipitation followed by sequencing (ULI-NChIP-seq) and explored the difference in H3K9ac occupancy between IVF/NF and SCNT embryos. We identified aberrantly acetylated regions (AARs) in SCNT embryos as an epigenetic barrier, which contain many genes critical to embryo development that are significantly downregulated or silenced in SCNT embryos. Although TSA treatment can largely fix most AARs and improve SCNT efficiency, its effects are restrained by the native epigenetic environments in donor cumulus cells. Intriguingly, we identified significant enrichment of the Dux motif in the regions with hyper-H3K9ac signals specifically in early 2C (e2C) IVF embryos. Dux is silenced in e2C SCNT embryos. Strikingly, injection of full-length Dux mRNA greatly restores H3K9ac occupancy in AARs, drives 2C activation, and improves SCNT efficiency in vitro and in vivo. Moreover, the Dux C-terminal domain is essential to fix aberrant acetylation and promote reprogramming. We also revealed that TSA and Kdm4b improved SCNT efficiency largely dependent on Dux. Our study identifies Dux as a crucial TF that improves SCNT efficiency through a more accurate epigenetic landscape modulation and provides a novel approach to improve mammalian cloning efficiency. In mice, ∼70% of SCNT embryos are arrested at early cleavage stages (Gao et al., 2018Gao R. Wang C. Gao Y. Xiu W. Chen J. Kou X. Zhao Y. Liao Y. Bai D. Qiao Z. et al.Inhibition of aberrant DNA re-methylation improves post-implantation development of somatic cell nuclear transfer embryos.Cell Stem Cell. 2018; 23: 426-435.e5Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar; Liu et al., 2016aLiu W. Liu X. Wang C. Gao Y. Gao R. Kou X. Zhao Y. Li J. Wu Y. Xiu W. et al.Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing.Cell Discov. 2016; 2: 16010Crossref PubMed Scopus (104) Google Scholar; Matoba et al., 2014Matoba S. Liu Y. Lu F. Iwabuchi K.A. Shen L. Inoue A. Zhang Y. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.Cell. 2014; 159: 884-895Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar; Ogura et al., 2013Ogura A. Inoue K. Wakayama T. Recent advancements in cloning by somatic cell nuclear transfer.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2013; 368: 20110329Crossref PubMed Scopus (131) Google Scholar). Our previous study demonstrated a defective global H3K9ac modification in SCNT embryos compared to ICSI embryos, whereas TSA treatment can efficiently fix this defect, especially from the 1C to the 2C stage (Wang et al., 2007Wang F. Kou Z. Zhang Y. Gao S. Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos.Biol. Reprod. 2007; 77: 1007-1016Crossref PubMed Scopus (141) Google Scholar). However, the potential mechanism by which H3K9ac occupancy improves reprogramming efficiency in SCNT remains elusive. To address this, we used ULI-NChIP-seq to generate a genome-wide landscape of H3K9ac signal during early embryo development (Figure 1A). The global H3K9ac profiles are highly correlated between ULI-NChIP-seq and the ENCODE project using massive cells (Figure S1A). Moreover, ULI-NChIP-seq results using as few as 200 cells fully recapitulate the H3K9ac peaks in the ENCODE project (Figure S1B). The H3K9ac signal was highly reproducible between the replicates (Figure S1C). In accordance with our previous study, we observed a global hypo-H3K9ac signal in SCNT embryos, whereas TSA treatment quickly and significantly increased genome-wide H3K9ac. However, after TSA removal, the level of H3K9ac in SCNT embryos declined and was still lower than that of IVF embryos at the 2C stage (Figures S1D–S1G). We then scanned the genome with a 5-kb sliding window to identify regions with aberrant H3K9ac signal intensity in SCNT embryos compared to IVF/NF embryos at each developmental stage. The regions with aberrantly high H3K9ac signals in SCNT embryos are termed aberrantly highly acetylated regions (AHARs). Similarly, the regions with aberrantly low H3K9ac signals in SCNT embryos are termed aberrantly low acetylated regions (ALARs). AHARs and ALARs together make up the AARs (refer to Data S1). The number of AHARs and ALARs precipitously decreased from 1C SCNT embryos to morula embryos (Figures 1B and 1C). Consistently, ALARs covered more genomic regions than AHARs in the first two cell cycles, which was in line with the immunostaining results (Figures S1D–S1G and S2A). Of note, both ALARs and AHARs accounted for a small fraction of the genome in late 2C (l2C) and morula embryos (Figure S2A). These findings together imply that these AARs are successfully reprogrammed by the l2C stage as long as SCNT embryos develop to the l2C and later stages. Alternatively, SCNT embryos with many AARs might fail to develop to the l2C or later stages. It was reported that H3K9me3 was enriched in reprogramming-resistant regions (RRRs) in donor somatic cells (Matoba et al., 2014Matoba S. Liu Y. Lu F. Iwabuchi K.A. Shen L. Inoue A. Zhang Y. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.Cell. 2014; 159: 884-895Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Consistent with that, H3K9ac in RRRs was significantly lower in early SCNT embryos than in IVF embryos (Figure S2B). Intriguingly, AHARs were significantly enriched for H3K9ac and DNase I signals but depleted for H3K9me3 signals in donor cumulus cells compared to ALARs (Figure 1D; Djekidel et al., 2018Djekidel M.N. Inoue A. Matoba S. Suzuki T. Zhang C. Lu F. Jiang L. Zhang Y. Reprogramming of chromatin accessibility in somatic cell nuclear transfer is DNA replication independent.Cell Rep. 2018; 23: 1939-1947Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This indicates that chromatin states of donor cells have an important impact on AARs in SCNT embryos. Further analysis found that most AARs in 2C embryos were inherited from 1C embryos and resisted reprogramming. In contrast, only ∼37% of AARs in morula embryos were inherited from 1C embryos (Figure S2C). This suggested that a majority of inherited AARs were faithfully remodeled in successfully reprogrammed morula embryos, whereas new AARs formed as well during the first cell fate determination at the morula stage (Figures 1B and S2C). Particularly, the H3K9ac signal in X chromosome inactivation center (XIC) was precipitously increased from 1C to morula SCNT embryos and became significantly higher than that in morula NF embryos (Figures 1E and S2D). In contrast, H3K9ac signal levels in X chromosomes were similar between SCNT and NF morula embryos (Figure S2D). These results clearly demonstrated that the aberrant increase in H3K9ac signal at Xist loci in SCNT embryos is not an artifact from the X dose difference between SCNT embryos (XX donor cumulus cells) and NF embryos (both XX and XY). Together, these results suggested that SCNT embryos showed abnormalities in H3K9ac signal before entering differentiation, which might account for the low blastocyst rate and further influence implantation. To investigate the potential function of aberrant H3K9ac in the temporal regulation of gene expression during early embryo development, we calculated and compared H3K9ac levels in the promoters of different gene sets between IVF/NF and SCNT embryos. Strikingly, H3K9ac levels in the promoters of the 2C-specific genes were significantly lower in SCNT embryos than in IVF embryos throughout the 2C stage (Figure S2E). Interestingly, the expression levels of genes within AHARs were greatly increased in SCNT embryos compared to IVF/NF embryos. The pattern was opposite for genes within ALARs (Figures S2F and S2G). This finding suggests that aberrant histone H3K9ac is an important epigenetic impediment for appropriate 2C gene activation. TSA, an HDAC inhibitor, has been widely utilized to improve SCNT efficiency (Akagi et al., 2011Akagi S. Matsukawa K. Mizutani E. Fukunari K. Kaneda M. Watanabe S. Takahashi S. Treatment with a histone deacetylase inhibitor after nuclear transfer improves the preimplantation development of cloned bovine embryos.J. Reprod. Dev. 2011; 57: 120-126Crossref PubMed Scopus (47) Google Scholar; Kishigami et al., 2006Kishigami S. Mizutani E. Ohta H. Hikichi T. Thuan N.V. Wakayama S. Bui H.T. Wakayama T. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.Biochem. Biophys. Res. Commun. 2006; 340: 183-189Crossref PubMed Scopus (461) Google Scholar; Rybouchkin et al., 2006Rybouchkin A. Kato Y. Tsunoda Y. Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer.Biol. Reprod. 2006; 74: 1083-1089Crossref PubMed Scopus (239) Google Scholar; Wang et al., 2007Wang F. Kou Z. Zhang Y. Gao S. Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos.Biol. Reprod. 2007; 77: 1007-1016Crossref PubMed Scopus (141) Google Scholar; Zhao et al., 2010Zhao J. Hao Y. Ross J.W. Spate L.D. Walters E.M. Samuel M.S. Rieke A. Murphy C.N. Prather R.S. Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos.Cell. Reprogram. 2010; 12: 75-83Crossref PubMed Scopus (120) Google Scholar). In order to understand how TSA improves reprogramming efficiency, we first profiled gene expression changes in SCNT embryos upon TSA treatment. There were a few hundred differentially expressed genes between l2C SCNT embryos treated with and without TSA (Figure S3A). This is consistent with a previous study showing limited effects on gene expression in SCNT embryos upon TSA treatment (Inoue et al., 2015Inoue K. Oikawa M. Kamimura S. Ogonuki N. Nakamura T. Nakano T. Abe K. Ogura A. Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer.Sci. Rep. 2015; 5: 10127Crossref PubMed Scopus (24) Google Scholar). Furthermore, TSA showed heterogeneity in gene recovery. For example, TSA only fully rescued some genes, whereas others, such as Sall4 and Ezh2, exhibited partial rescue. However, certain genes essential for preimplantation development failed to be rescued, such as the 2C gene Zscan4d (Figure S3B; Eckersley-Maslin et al., 2016Eckersley-Maslin M.A. Svensson V. Krueger C. Stubbs T.M. Giehr P. Krueger F. Miragaia R.J. Kyriakopoulos C. Berrens R.V. Milagre I. et al.MERVL/Zscan4 network activation results in transient genome-wide DNA demethylation of mESCs.Cell Rep. 2016; 17: 179-192Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar; Falco et al., 2007Falco G. Lee S.-L. Stanghellini I. Bassey U.C. Hamatani T. Ko M.S.H. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells.Dev. Biol. 2007; 307: 539-550Crossref PubMed Scopus (155) Google Scholar; Zalzman et al., 2010Zalzman M. Falco G. Sharova L.V. Nishiyama A. Thomas M. Lee S.-L. Stagg C.A. Hoang H.G. Yang H.-T. Indig F.E. et al.Zscan4 regulates telomere elongation and genomic stability in ES cells.Nature. 2010; 464: 858-863Crossref PubMed Scopus (251) Google Scholar). TSA treatment results in extensive changes in the H3K9ac landscape in SCNT embryos and could greatly increase the development potential of SCNT embryos (Figures S1D–S1G, S2A, and S2B; Table S1). Therefore, we next examined how TSA altered H3K9ac occupancy in the AARs in SCNT embryos. Surprisingly, TSA quickly and greatly restored H3K9ac levels in most AARs to levels resembling those of IVF embryos (Figures 2C and S3C). Of note, there still existed certain regions in which the aberrant H3K9ac signal in SCNT embryos failed to be fixed by TSA treatment (Figures" @default.
• W3092364740 created "2020-10-15" @default.
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• W3092364740 date "2021-01-01" @default.
• W3092364740 modified "2023-10-02" @default.
• W3092364740 title "Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer" @default.
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• W3092364740 doi "https://doi.org/10.1016/j.stem.2020.09.006" @default.

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