, Volume 70, Issue 5, pp 1469–1477 | Cite as

Treatment of donor cells with recombinant KDM4D protein improves preimplantation development of cloned ovine embryos

  • Yumei Zhang
  • Qianqian Wang
  • Kailing Liu
  • Enen Gao
  • Hong Guan
  • Jian HouEmail author
Short Communication


Incomplete epigenetic reprogramming is one of the major factors affecting the development of embryos cloned by somatic cell nuclear transfer (SCNT). Histone 3 lysine 9 (H3K9) trimethylation has been identified as a key barrier to efficient reprogramming by SCNT. The aim of this study was to explore a method of downregulating H3K9me3 levels in donor cells by using histone lysine demethylase (KDM) protein. When sheep fetal fibroblast cells were treated with recombinant human KDM4D protein (rhKDM4D), the levels of H3K9 trimethylation and dimethylation were both significantly decreased. After SCNT, rhKDM4D-treated donor cells supported significantly higher percentage of cloned embryos developing into blastocysts as compared to non-treated control cells. Moreover, the blastocyst quality was also improved by rhKDM4D treatment of donor cells, as assessed by the total cell number in blastocysts and the expression of developmental genes including SOX2, NANOG and CDX2. These results indicate that treatment of donor cells with recombinant KDM4D protein can downregulate the levels of H3K9 trimethylation and dimethylation and improve the developmental competence of SCNT embryos. This strategy may be convenient to be used in KDM4-assisted SCNT procedure for improving the efficiency of cloning.


H3K9 methylation KDM4D Sheep Somatic cell nuclear transfer 



This work was supported by the National Natural Science Foundation of China (Grant No. 31172208) and China Agriculture Research System (Grant No. CARS-39-04).

Compliance with ethical standards

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Supplementary material

10616_2018_224_MOESM1_ESM.pdf (293 kb)
Supplementary material 1 (PDF 292 kb)


  1. Antony J, Oback F, Chamley LW, Oback B, Laible G (2013) Transient JMJD2B-mediated reduction of H3K9me3 levels improves reprogramming of embryonic stem cells into cloned embryos. Mol Cell Biol 33:974–983. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Cao Z, Li Y, Chen Z, Wang H, Zhang M, Zhou N, Wu R, Ling Y, Fang F, Li N, Zhang Y (2015) Genome-wide dynamic profiling of histone methylation during nuclear transfer-mediated porcine somatic cell reprogramming. PLoS ONE 10:e0144897. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chen J, Liu H, Liu J, Qi J, Wei B, Yang J, Liang H, Chen Y, Chen J, Wu Y, Guo L, Zhu J, Zhao X, Peng T, Zhang Y, Chen S, Li X, Li D, Wang T, Pei D (2013) H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat Genet 45:34–42. CrossRefPubMedGoogle Scholar
  4. Chung YG, Matoba S, Liu Y, Eum JH, Lu F, Jiang W, Lee JE, Sepilian V, Cha KY, Lee DR, Zhang Y (2015) Histone demethylase expression enhances human somatic cell nuclear transfer efficiency and promotes derivation of pluripotent stem cells. Cell Stem Cell 17:758–766. CrossRefPubMedGoogle Scholar
  5. Fu L, Zhang J, Yan FX, Guan H, An XR, Hou J (2012) Abnormal histone H3K9 dimethylation but normal dimethyltransferase EHMT2 expression in cloned sheep embryos. Theriogenology 78:1929–1938. CrossRefPubMedGoogle Scholar
  6. Fu L, Yan FX, An XR, Hou J (2014) Effects of the histone methyltransferase inhibitor UNC0638 on histone H3K9 dimethylation of cultured ovine somatic cells and development of resulting early cloned embryos. Reprod Domest Anim 49:e21–e25. CrossRefPubMedGoogle Scholar
  7. Hou J, Liu L, Zhang J, Cui XH, Yan FX, Guan H, Chen YF, An XR (2008) Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation. BMC Dev Biol 8:60. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Huang J, Zhang H, Yao J, Qin G, Wang F, Wang X, Luo A, Zheng Q, Cao C, Zhao J (2016) BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei. Reproduction 151:39–49. CrossRefPubMedGoogle Scholar
  9. Huang Y, Jiang X, Yu M, Huang R, Yao J, Li M, Zheng F, Yang X (2017) Beneficial effects of diazepin–quinazolin–amine derivative (BIX-01294) on preimplantation development and molecular characteristics of cloned mouse embryos. Reprod Fertil Dev 29:1260–1269. CrossRefPubMedGoogle Scholar
  10. Kallingappa PK, Turner PM, Eichenlaub MP, Green AL, Oback FC, Chibnall AM, Wells DN, Oback B (2016) Quiescence loosens epigenetic constraints in bovine somatic cells and improves their reprogramming into totipotency. Biol Reprod 95:16. CrossRefPubMedGoogle Scholar
  11. Krishnan S, Trievel RC (2013) Structural and functional analysis of JMJD2D reveals molecular basis for site-specific demethylation among JMJD2 demethylases. Structure 21:98–108. CrossRefPubMedGoogle Scholar
  12. Liu W, Liu X, Wang C, Gao Y, Gao R, Kou X, Zhao Y, Li J, Wu Y, Xiu W, Wang S, Yin J, Liu W, Cai T, Wang H, Zhang Y, Gao S (2016) Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing. Cell Discov 2:16010. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Liu Z, Cai Y, Wang Y, Nie Y, Zhang C, Xu Y, Zhang X, Lu Y, Wang Z, Poo M, Sun Q (2018) Cloning of macaque monkeys by somatic cell nuclear transfer. Cell 172:1–7. CrossRefGoogle Scholar
  14. Loh YH, Zhang W, Chen X, George J, Ng HH (2007) Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. Gene Dev 21:2545–2557. CrossRefPubMedGoogle Scholar
  15. Matoba S, Liu Y, Lu F, Iwabuchi KA, Shen L, Inoue A, Zhang Y (2014) Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell 159:884–895. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Ruan D, Peng J, Wang X, Ouyang Z, Zou Q, Yang Y, Chen F, Ge W, Wu H, Liu Z, Zhao Y, Zhao B, Zhang Q, Lai C, Fan N, Zhou Z, Liu Q, Li N, Jin Q, Shi H, Xie J, Song H, Yang X, Chen J, Wang K, Li X, Lai L (2018) XIST derepression in active X chromosome hinders pig somatic cell nuclear transfer. Stem Cell Rep 10:494–508. CrossRefGoogle Scholar
  17. Santos F, Zakhartchenko V, Stojkovic M, Peters A, Jenuwein T, Wolf E, Reik W, Dean W (2003) Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol 13:1116–1121CrossRefGoogle Scholar
  18. Sridharan R, Gonzales-Cope M, Chronis C, Bonora G, McKee R, Huang C, Patel S, Lopez D, Mishra N, Pellegrini M, Carey M, Garcia BA, Plath K (2013) Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1gamma in reprogramming to pluripotency. Nat Cell Biol 15:872–882. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Wang Y, Zhang Y, Mao T, Yan B, Deng R, Wei B, Zhang Y, Liu J (2017) Treatment donor cells with UNC0638 modify the abnormal histone H3K9 dimethylation and gene expression in cloned goat embryos. Small Rumin Res 156:27–32. CrossRefGoogle Scholar
  20. Wei J, Antony J, Meng F, MacLean P, Rhind R, Laible G, Oback B (2017) KDM4B-mediated reduction of H3K9me3 and H3K36me3 levels improves somatic cell reprogramming into pluripotency. Sci Rep 7:7514. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Whetstine JR, Nottke A, Lan F, Huarte M, Smolikov S, Chen Z, Spooner E, Li E, Zhang G, Colaiacovo M, Shi Y (2006) Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell 125:467–481. CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Agrobiotechnology, College of Biological ScienceChina Agricultural UniversityBeijingChina

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