GSK-J4-Mediated Transcriptomic Alterations in Differentiating Embryoid Bodies

  • Mandal, Chanchal (Department of Molecular and Life Science, Hanyang University) ;
  • Kim, Sun Hwa (Department of Molecular and Life Science, Hanyang University) ;
  • Kang, Sung Chul (Department of Molecular and Life Science, Hanyang University) ;
  • Chai, Jin Choul (Department of Molecular and Life Science, Hanyang University) ;
  • Lee, Young Seek (Department of Molecular and Life Science, Hanyang University) ;
  • Jung, Kyoung Hwa (Institute of Natural Science and Technology, Hanyang University) ;
  • Chai, Young Gyu (Department of Molecular and Life Science, Hanyang University)
  • Received : 2017.05.09
  • Accepted : 2017.08.20
  • Published : 2017.10.31


Histone-modifying enzymes are key players in the field of cellular differentiation. Here, we used GSK-J4 to profile important target genes that are responsible for neural differentiation. Embryoid bodies were treated with retinoic acid ($10{\mu}M$) to induce neural differentiation in the presence or absence of GSK-J4. To profile GSKJ4-target genes, we performed RNA sequencing for both normal and demethylase-inhibited cells. A total of 47 and 58 genes were up- and down-regulated, respectively, after GSK-J4 exposure at a log2-fold-change cut-off value of 1.2 (p-value < 0.05). Functional annotations of all of the differentially expressed genes revealed that a significant number of genes were associated with the suppression of cellular proliferation, cell cycle progression and induction of cell death. We also identified an enrichment of potent motifs in selected genes that were differentially expressed. Additionally, we listed upstream transcriptional regulators of all of the differentially expressed genes. Our data indicate that GSK-J4 affects cellular biology by inhibiting cellular proliferation through cell cycle suppression and induction of cell death. These findings will expand the current understanding of the biology of histone-modifying enzymes, thereby promoting further investigations to elucidate the underlying mechanisms.


Supported by : National Research Foundation of Korea (NRF)


  1. Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114- 2120.
  2. Burgold, T., Spreafico, F., De Santa, F., Totaro, M.G., Prosperini, E., Natoli, G., and Testa, G. (2008). The histone H3 lysine 27-specific demethylase Jmjd3 is required for neural commitment. PLoS One 3, e3034.
  3. Chen, C., Chang, Y.C., Lan, M.S., and Breslin, M. (2013). Leptin stimulates ovarian cancer cell growth and inhibits apoptosis by increasing cyclin D1 and Mcl-1 expression via the activation of the MEK/ERK1/2 and PI3K/Akt signaling pathways. Int. J. Oncol. 42, 1113-1119.
  4. Chen, Y., Jiang, J., Zhao, M., Luo, X., Liang, Z., Zhen, Y., Fu, Q., Deng, X., Lin, X., Li, L., et al. (2016). microRNA-374a suppresses colon cancer progression by directly reducing CCND1 to inactivate the PI3K/AKT pathway. Oncotarget 7, 41306-41319.
  5. Cho, H.J., Park, S.M., Hwang, E.M., Baek, K.E., Kim, I.K., Nam, I.K., Im, M.J., Park, S.H., Bae, S., Park, J.Y., et al. (2010). Gadd45b mediates Fas-induced apoptosis by enhancing the interaction between p38 and retinoblastoma tumor suppressor. J. Biol. Chem. 285, 25500-25505.
  6. Cloos, P.A., Christensen, J., Agger, K., Maiolica, A., Rappsilber, J., Antal, T., Hansen, K.H., and Helin, K. (2006). The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature 442, 307-311.
  7. Dai, J., Wei, R.J., Li, R., Feng, J.B., Yu, Y.L., and Liu, P.S. (2016). A study of CCND1 with epithelial ovarian cancer cell proliferation and apoptosis. Eur. Rev. Med. Pharmacol. Sci. 20, 4230-4235.
  8. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21.
  9. Ferreri, A.J., Illerhaus, G., Zucca, E., and Cavalli, F. (2010). Flows and flaws in primary central nervous system lymphoma. Nat. Rev. Clin. Oncol. 7, doi:10 1038/nrclinonc 2010 1039-c1031; author reply doi:1010:1038/nrclinonc 2010 1039-c1032.
  10. Flores, O., and Burnstein, K.L. (2010). GADD45gamma: a new vitamin D-regulated gene that is antiproliferative in prostate cancer cells. Endocrinology 151, 4654-4664.
  11. Greenberg, V.L., Williams, J.M., Cogswell, J.P., Mendenhall, M., and Zimmer, S.G. (2001). Histone deacetylase inhibitors promote apoptosis and differential cell cycle arrest in anaplastic thyroid cancer cells. Thyroid 11, 315-325.
  12. Guo, W., Zhu, T., Dong, Z., Cui, L., Zhang, M., and Kuang, G. (2013). Decreased expression and aberrant methylation of Gadd45G is associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma. Clin. Exp. Metastasis 30, 977-992.
  13. Heinemann, B., Nielsen, J.M., Hudlebusch, H.R., Lees, M.J., Larsen, D.V., Boesen, T., Labelle, M., Gerlach, L.O., Birk, P. and Helin, K. (2014). Inhibition of demethylases by GSK-J1/J4. Nature 514, E1-2.
  14. Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y.C., Laslo, P., Cheng, J.X., Murre, C., Singh, H., and Glass, C.K. (2010). Simple combinations of lineage-determining transcription factors prime cisregulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576-589.
  15. Hofstetter, C., Kampka, J.M., Huppertz, S., Weber, H., Schlosser, A., Muller, A.M., and Becker, M. (2016). Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage. J. Cell Sci. 129, 788-803.
  16. Hong, S., Cho, Y.W., Yu, L.R., Yu, H., Veenstra, T.D., and Ge, K. (2007). Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases. Proc. Natl. Acad. Sci. USA 104, 18439-18444.
  17. Huang da, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-57.
  18. Ishida, K., Yuge, Y., Hanaoka, M., Yasukawa, M., Minami, Y., Ogawa, M., Masumoto, K.H., Shigeyoshi, Y., Saito, M., and Tsuji, T. (2013). Gadd45g regulates dental epithelial cell proliferation through p38 MAPK-mediated p21 expression. Genes Cells 18, 660-671.
  19. Jiang, P., Rao, E.Y., Meng, N., Zhao, Y., and Wang, J.J. (2010). MicroRNA-17-92 significantly enhances radioresistance in human mantle cell lymphoma cells. Radiat Oncol. 5, 100.
  20. Kageyama, K., Sugiyama, A., Murasawa, S., Asari, Y., Niioka, K., Oki, Y., and Daimon, M. (2015). Aphidicolin inhibits cell proliferation via the p53-GADD45beta pathway in AtT-20 cells. Endocr J. 62, 645- 654.
  21. Kamikawa, Y.F., and Donohoe, M.E. (2015). Histone demethylation maintains Prdm14 and Tsix expression and represses xIst in embryonic stem cells. PLoS One 10, e0125626.
  22. Kang, S.C., Kim, S.K., Chai, J.C., Kim, S.H., Won, K.J., Lee, Y.S., Jung, K.H., and Chai, Y.G. (2015). Transcriptomic profiling and H3K27me3 distribution reveal both demethylase-dependent and independent regulation of developmental gene transcription in cell differentiation. PLoS One 10, e0135276.
  23. Kapral, M., Strzalka-Mrozik, B., Kowalczyk, M., Paluch, J., Gola, J., Gierek, T., and Weglarz, L. (2011). Transcriptional activities of histone H3, cyclin D1 and claudin 7 encoding genes in laryngeal cancer. Eur. Arch. Otorhinolaryngol. 268, 709-714.
  24. Kim, J.H., and Workman, J.L. (2010). Histone acetylation in heterochromatin assembly. Genes Dev. 24, 738-740.
  25. Kramer, A., Green, J., Pollard, J., Jr., and Tugendreich, S. (2014). Causal analysis approaches in ingenuity pathway analysis. Bioinformatics 30, 523-530.
  26. Kruidenier, L., Chung, C.W., Cheng, Z., Liddle, J., Che, K., Joberty, G., Bantscheff, M., Bountra, C., Bridges, A., Diallo, H., et al. (2012). A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 488, 404-408.
  27. Lai, J.P., Sandhu, D.S., Yu, C., Moser, C.D., Hu, C., Shire, A.M., Aderca, I., Murphy, L.M., Adjei, A.A., Sanderson, S., et al. (2010). Sulfatase 2 protects hepatocellular carcinoma cells against apoptosis induced by the PI3K inhibitor LY294002 and ERK and JNK kinase inhibitors. Liver Int. 30, 1522-1528.
  28. Lane, A.A., Chapuy, B., Lin, C.Y., Tivey, T., Li, H., Townsend, E.C., van Bodegom, D., Day, T.A., Wu, S.C., Liu, H., et al. (2014). Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 Lys27 trimethylation. Nat. Genet. 46, 618-623.
  29. Lee, J.S., Smith, E., and Shilatifard, A. (2010). The language of histone crosstalk. Cell 142, 682-685.
  30. Li, B., Carey, M., and Workman, J.L. (2007). The role of chromatin during transcription. Cell 128, 707-719.
  31. Li, Y.L., Wang, J., Zhang, C.Y., Shen, Y.Q., Wang, H.M., Ding, L., Gu, Y.C., Lou, J.T., Zhao, X.T., Ma, Z.L., et al. (2016). MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2. Oncotarget 7, 59287-59298.
  32. Liu, L., Tran, E., Zhao, Y., Huang, Y., Flavell, R., and Lu, B. (2005). Gadd45 beta and Gadd45 gamma are critical for regulating autoimmunity. J. Exp. Med. 202, 1341-1347.
  33. Liu, B., Zhang, Y.H., Jiang, Y., Li, L.L., Chen, Q., He, G.Q., Tan, X.D., and Li, C.Q. (2015a). Gadd45b is a novel mediator of neuronal apoptosis in ischemic stroke. Int. J. Biol. Sci. 11, 353-360.
  34. Liu, Z., Cao, W., Xu, L., Chen, X., Zhan, Y., Yang, Q., Liu, S., Chen, P., Jiang, Y., Sun, X., et al. (2015b). The histone H3 lysine-27 demethylase Jmjd3 plays a critical role in specific regulation of Th17 cell differentiation. J. Mol. Cell Biol. 7, 505-516.
  35. Love, M.I., Huber, W. and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.
  36. Maes, T., Carceller, E., Salas, J., Ortega, A. and Buesa, C. (2015). Advances in the development of histone lysine demethylase inhibitors. Curr. Opin. Pharmacol. 23, 52-60.
  37. Mak, S.K. and Kultz, D. (2004). Gadd45 proteins induce G2/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress. J. Biol. Chem. 279, 39075-39084.
  38. Mandal, C., Kim, S.H., Chai, J.C., Oh, S.M., Lee, Y.S., Jung, K.H. and Chai, Y.G. (2016). RNA Sequencing reveals the alteration of the expression of novel genes in ethanol-treated embryoid bodies. PLoS One 11, e0149976.
  39. Morales Torres, C., Laugesen, A. and Helin, K. (2013). Utx is required for proper induction of ectoderm and mesoderm during differentiation of embryonic stem cells. PLoS One 8, e60020.
  40. Nettersheim, D., Jostes, S., Fabry, M., Honecker, F., Schumacher, V., Kirfel, J., Kristiansen, G. and Schorle, H. (2016). A signaling cascade including ARID1A, GADD45B and DUSP1 induces apoptosis and affects the cell cycle of germ cell cancers after romidepsin treatment. Oncotarget 7, 74931-74946.
  41. Rui, L., Emre, N.C., Kruhlak, M.J., Chung, H.J., Steidl, C., Slack, G., Wright, G.W., Lenz, G., Ngo, V.N., Shaffer, A.L., et al. (2010). Cooperative epigenetic modulation by cancer amplicon genes. Cancer Cell. 18, 590-605.
  42. Ruijtenberg, S. and van den Heuvel, S. (2016). Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression. Cell Cycle. 15, 196- 212.
  43. Sakaki, H., Okada, M., Kuramoto, K., Takeda, H., Watarai, H., Suzuki, S., Seino, S., Seino, M., Ohta, T., Nagase, S., et al. (2015). GSKJ4, a selective Jumonji H3K27 demethylase inhibitor, effectively targets ovarian cancer stem cells. Anticancer Res. 35, 6607-6614.
  44. Salvador, J.M., Brown-Clay, J.D. and Fornace, A.J., Jr. (2013). Gadd45 in stress signaling, cell cycle control, and apoptosis. Adv. Exp. Med. Biol. 793, 1-19.
  45. Sandhu, S.K., Fassan, M., Volinia, S., Lovat, F., Balatti, V., Pekarsky, Y. and Croce, C.M. (2013). B-cell malignancies in microRNA Emu-miR- 17-92 transgenic mice. Proc. Natl. Acad. Sci. USA 110, 18208- 18213.
  46. Smith, S. and Watters, J. (2014). Role of JMJD3 and histone modifications in intermittent hypoxia-induced neuroinflammation (1120.6). FASEB J. 28, 1120.1126.
  47. Tennant, B.R., Hurley, P., Dhillon, J., Gill, A., Whiting, C. and Hoffman, B.G. (2015). The TrxG complex mediates cytokine induced de novo enhancer formation in islets. PLoS One. 10, e0141470.
  48. Vairapandi, M., Balliet, A.G., Hoffman, B. and Liebermann, D.A. (2002). GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress. J. Cell Physiol. 192, 327-338.
  49. Vaquerizas, J.M., Kummerfeld, S.K., Teichmann, S.A., and Luscombe, N.M. (2009). A census of human transcription factors: function, expression and evolution. Nat Rev Genet. 10, 252-263.
  50. Wang, X.W., Zhan, Q., Coursen, J.D., Khan, M.A., Kontny, H.U., Yu, L., Hollander, M.C., O'Connor, P.M., Fornace, A.J., Jr., and Harris, C.C. (1999). GADD45 induction of a G2/M cell cycle checkpoint. Proc. Natl. Acad. Sci. USA 96, 3706-3711.
  51. Wang, N., Wei, H., Yin, D., Lu, Y., Zhang, Y., Jiang, D., Jiang, Y., and Zhang, S. (2014). Cyclin D1b overexpression inhibits cell proliferation and induces cell apoptosis in cervical cancer cells in vitro and in vivo. Int. J. Clin. Exp. Pathol. 7, 4016-4023.
  52. Watarai, H., Okada, M., Kuramoto, K., Takeda, H., Sakaki, H., Suzuki, S., Seino, S., Oizumi, H., Sadahiro, M., and Kitanaka, C. (2016). Impact of H3K27 Demethylase Inhibitor GSKJ4 on NSCLC Cells Alone and in Combination with Metformin. Anticancer Res. 36, 6083-6092.
  53. Weiss, W.A., Taylor, S.S., and Shokat, K.M. (2007). Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat. Chem. Biol. 3, 739-744.
  54. Wissmann, M., Yin, N., Muller, J.M., Greschik, H., Fodor, B.D., Jenuwein, T., Vogler, C., Schneider, R., Gunther, T., Buettner, R., et al. (2007). Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression. Nat. Cell. Biol. 9, 347-353.
  55. Xiang, Y., Zhu, Z., Han, G., Lin, H., Xu, L., and Chen, C.D. (2007). JMJD3 is a histone H3K27 demethylase. Cell Res. 17, 850-857.
  56. Xiao, C., Srinivasan, L., Calado, D.P., Patterson, H.C., Zhang, B., Wang, J., Henderson, J.M., Kutok, J.L., and Rajewsky, K. (2008). Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat. Immunol. 9, 405-414.
  57. Xie, B., Zhang, H., Wei, R., Li, Q., Weng, X., Kong, Q., and Liu, Z. (2016). Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming. Reproduction 151, 9-16.
  58. Yang, S., Sun, H.Y., Xiao, F.J., Li, Q.F., Xu, J., Guo, Z.K., Wang, H.X., and Wang, L.S. (2014). [Effect of microRNA-17-92 cluster on the biological characteristics of K562 cells and its mechanisms]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 22, 20-24.
  59. Zhang, Y., Ueno, Y., Liu, X.S., Buller, B., Wang, X., Chopp, M., and Zhang, Z.G. (2013). The MicroRNA-17-92 cluster enhances axonal outgrowth in embryonic cortical neurons. J. Neurosci. 33, 6885-6894.
  60. Zhao, L., Gu, H., Chang, J., Wu, J., Wang, D., Chen, S., Yang, X. and Qian, B. (2014). MicroRNA-383 regulates the apoptosis of tumor cells through targeting Gadd45g. PLoS One 9, e110472.
  61. Zhu, J., Sen, S., Wei, C., and Frazier, M.L. (2010). Cyclin D1b represses breast cancer cell growth by antagonizing the action of cyclin D1a on estrogen receptor alpha-mediated transcription. Int. J. Oncol. 36, 39-48.