Skip to main content

Knocking down of UTX in NCCIT cells enhance cell attachment and promote early neuronal cell differentiation

Abstract

Neural differentiation involves complex changes of gene expression patterns, which are controlled by chromatin remodeling that promotes or inhibits neurogenesis and gliogenesis. To study the roles of the ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) during neuronal differentiation, we performed gene expression analysis in gene knock down experiments using an artificial miRNA technique. Microarray analysis found that a total of 919 genes were differentially altered in the UTX-KD embryonic carcinoma (NCCIT) cells, and a total of 964 genes in the UTX-KD embryoid bodies (EBs) by 2.0 fold cut off value. Gene ontology analysis revealed the association of cell adhesion related genes were enhanced by UTX-KD. Morphological analysis also showed more attached neurites during differentiation with UTX-KD cells. Differentiated neurons were characterized as GABAergic neurons expressing typical neuronal markers, TU-20 and GAD65. Collectively, our data suggest that knocking down of UTX enhances cell attachment by enhancing related gene expressions and thereby promotes early neuronal cell differentiation.

This is a preview of subscription content, access via your institution.

References

  1. Tyssowski, K., Kishi, Y. & Gotoh, Y. Chromatin regulators of neural development. Neuroscience 264, 4–16 (2014).

    CAS  Article  Google Scholar 

  2. Feng, J., Fouse, S. & Fan, G. Epigenetic regulation of neural gene expression and neuronal function. Pediatr. Res. 61, 58R-63R (2007).

    Google Scholar 

  3. Hsieh, J. & Gage, F.H. Chromatin remodeling in neural development and plasticity. Curr. Opin. Cell. Biol. 17, 664–671 (2005).

    CAS  Article  Google Scholar 

  4. Wu, H. & Sun, Y.E. Epigenetic regulation of stem celldifferentiation. Pediatr. Res. 59, 21R-25R(2006).

    Article  Google Scholar 

  5. Chi, P., Allis, C.D. & Wang, G.G. Covalent histone modifications—miswritten, misinterpreted and miserased in human cancers. Nature reviews. Cancer 10, 457–469(2010).

    CAS  Article  Google Scholar 

  6. Jenuwein, T. & Allis, C.D. Translating the histone code. Science 293, 1074–1080(2001).

    CAS  Article  Google Scholar 

  7. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).

    CAS  Article  Google Scholar 

  8. Tammen, S.A., Friso, S. & Choi, S.W. Epigenetics: the link between nature and nurture. Molecular aspects of medicine 34, 753–764(2013).

    CAS  Article  Google Scholar 

  9. Mikkelsen, T.S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007).

    CAS  Article  Google Scholar 

  10. Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B. & Cavalli, G. Genome regulation by polycomb and trithorax proteins. Cell 128, 735–745 (2007).

    CAS  Article  Google Scholar 

  11. Trojer, P. & Reinberg, D. Histone lysine demethylases and their impact on epigenetics. Cell 125, 213–217 (2006).

    CAS  Article  Google Scholar 

  12. Welstead, G.G. et al. X-linked H3K27me3 demethylase Utx is required for embryonic development in a sexspecific manner. Proceedings of the National Academy of Sciences of the United States of America 109, 13004–13009(2012).

    CAS  Article  Google Scholar 

  13. Hong, S. et al. Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases. Proceedings of the National Academy of Sciences of the United States of America 104, 18439–18444 (2007).

    CAS  Article  Google Scholar 

  14. Boyer, L.A. et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349–353 (2006).

    CAS  Article  Google Scholar 

  15. Lee, T.I. et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301–313(2006).

    CAS  Article  Google Scholar 

  16. Van der Meulen, J., Speleman, F. & Van Vlierberghe, P. The H3K27me3 demethylase UTX in normal development and disease. Epigenetics 9, 658–668 (2014).

    Article  Google Scholar 

  17. Miller, S.A., Mohn, S.E. & Weinmann, A.S. Jmjd3 and UTX play a demethylase-independent role in chromatin remodeling to regulate T-box family memberdependent gene expression. Molecular cell 40, 594–605(2010).

    CAS  Article  Google Scholar 

  18. Damjanov, I., Horvat, B. & Gibas, Z. Retinoic acidinduced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT. Laboratory investigation; a journal of technical methods and pathology 68, 220–232 (1993).

    CAS  Google Scholar 

  19. Kim, S.K. et al. Human histone H3K79 methyltransferase DOT1L protein [corrected] binds actively transcribing RNA polymerase II to regulate gene expression. The Journal of biological chemistry 287, 39698–39709 (2012).

    CAS  Article  Google Scholar 

  20. Podrygajlo, G. et al. Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture. Cell and tissue research 336, 439–452 (2009).

    CAS  Article  Google Scholar 

  21. Donovan, P.J. & Gearhart, J. The end of the beginning for pluripotent stem cells. Nature 414, 92–97 (2001).

    CAS  Article  Google Scholar 

  22. Tegenge, M.A., Roloff, F. & Bicker, G. Rapid differentiation of human embryonal carcinoma stem cells (NT2) into neurons for neurite outgrowth analysis. Cell. Mol. Neurobiol. 31, 635–643 (2011).

    CAS  Article  Google Scholar 

  23. Frank, S.R., Schroeder, M., Fernandez, P., Taubert, S. & Amati, B. Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. Genes &development 15, 2069–2082(2001).

    CAS  Google Scholar 

  24. Jepsen, K. et al. SMRT-mediated repression of an H3K27 demethylase in progression from neural stem cell to neuron. Nature 450, 415–419 (2007).

    CAS  Article  Google Scholar 

  25. Seenundun, S. et al. UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis. The EMBO Journal 29, 1401–1411 (2010).

    CAS  Article  Google Scholar 

  26. Halder, D. et al. Chronic ethanol exposure increases goosecoid (GSC) expression in human embryonic carcinoma cell differentiation. J. Appl. Toxicol. 34, 66–75 (2014).

    CAS  Article  Google Scholar 

  27. Choi, M.R. et al. Ethanol-induced small heat shock protein genes in the differentiation of mouse embryonic neural stem cells. Archives of toxicology 85, 293–304 (2011).

    CAS  Article  Google Scholar 

  28. Baik, S.Y. et al. Fluoxetine-induced up-regulation of 14–3-3zeta and tryptophan hydroxylase levels in RBL-2H3 cells. Neuroscience letters 374, 53–57 (2005).

    CAS  Article  Google Scholar 

  29. Baek, M.N. et al. Artificial microRNA-based neurokinin-1 receptor gene silencing reduces alcohol consumption in mice. Neuroscience letters 475, 124–128 (2010).

    CAS  Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Young Gyu Chai.

Additional information

These authors contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mandal, C., Jung, K.H., Kang, S.C. et al. Knocking down of UTX in NCCIT cells enhance cell attachment and promote early neuronal cell differentiation. BioChip J 9, 182–193 (2015). https://doi.org/10.1007/s13206-015-9302-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13206-015-9302-4

Keywords

  • Neuronal differentiation
  • Embryonic carcinoma cells
  • Chromatin modification
  • UTX
  • Microarray analysis