Sains Malaysiana 46(10)(2017): 18871893

http://dx.doi.org/10.17576/jsm-2017-4610-27

 

AP4 Transcription Factor Binding Site is a Repressor Element in ek2 Promoter of Human Liver Carcinoma Cell Line, HepG2

(Tapak Pengikat Faktor Transkripsi AP4 adalah Unsur Penindas dalam Promoter ek2 Titisan Sel Karsinoma Hati Manusia, HepG2)

 

ZHI HUI TEH, CHEE SIAN KUAN, BOON HUAT LIM, WEI CUN SEE TOO & LING LING FEW*

 

School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

 

Received: 8 September 2016/Accepted: 6 March 2017

 

 

ABSTRACT

Ethanolamine kinase (EK) is the first enzyme in the Kennedy pathway for the biosynthesis of phosphatidylethanolamine. Although EK has been reported to be involved in phospholipid biosynthesis, carcinogenesis, cell growth, muscle development and sex determination during embryonic development, little is known about its transcriptional regulation by endogenous or exogenous signals. Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. Compared to ek1 gene, ek2 is expressed at a higher level in liver and EK2 isoforms also accept choline as substrate besides ethanolamine, which could contribute to liver carcinogenesis. The main aim of this study was to analyze and characterize the human ek2 promoter in cultured mammalian cells. Human ek2 (2011 bp) promoter was cloned into reporter vector, pGL4.10 [luc2] and the promoter activities were studied in human liver carcinoma (HepG2 cells). Sequence analyses showed that ek2 promoter contains numerous putative transcription factor binding sites including AP4 and it is devoid of a recognizable consensus TATA box but it contains a high number of guanine (G) and cytosine (C) nucleotides. PCR mutagenesis of three nucleotides at E-box motif of AP4 transcription binding site located between -293 and -276 of ek2 promoter was successfully performed to show that AP4 transcription factor binding site acts as a repressive element in the regulation of ek2 expression. AP4 upregulation has been implicated in bad prognosis of carcinoma, therefore the regulatory role of AP4 binding site reported in this study could be a link between ek2 and carcinogenesis. Although further studies need to be carried out to understand and to determine the repression mechanism of AP4 in ek2 promoter, the characterization and analysis of ek promoter performed in this study provide important understanding of its basal transcriptional regulation which would allow us to control ek expression levels in pathologic conditions that involve this gene.

 

Keywords: AP4; ethanolamine kinase; HepG2; promoter; transcription factor

ABSTRAK

Etanolamina kinase (EK) merupakan enzim pertama dalam laluan Kennedy untuk biosintesis fosfatidiletanolamina. Walaupun EK telah dilaporkan terlibat dalam biosintesis fosfolipid, karsinogenesis, pertumbuhan sel, perkembangan otot dan penentuan jantina semasa pertumbuhan embrio, maklumat tentang kawalan transkripsi EK oleh rangsangan dalaman atau luaran masih tidak jelas sehingga kini. EK manusia wujud sebagai isoform EK1, EKdan EK2β yang dikodkan oleh dua gen berasingan iaitu ek1 dan ek2. Berbanding dengan gen ek1, ek2 diekspres pada tahap yang lebih tinggi di dalam hati dan isoform EK2 juga menerima kolina sebagai substrat selain daripada etanolamina justeru menyumbang kepada karsinogenesis hati. Tujuan utama kajian ini adalah untuk menganalisis dan mencirikan promoter ek2 dalam sel mamalia. Promoter ek2 (2011 bp) manusia telah diklonkan ke dalam vektor pelapor pGL4.10 [luc2] dan dikaji dalam sel karsinoma hati manusia (HepG2). Analisis jujukan menunjukkan bahawa terdapat banyak tapak perlekatan bagi faktor transkripsi termasuk AP4 pada promoter ek2. Kajian ini juga menunjukkan tiada kehadiran kotak TATA yang boleh dikenali tetapi mengandungi nukleotida guanina (G) dan sistina (C) yang banyak. Mutagenesis PCR tiga nukleotida pada motif E-box dalam tapak perlekatan faktor transkripsi AP4 yang terletak antara -276 hingga -293 pada promoter ek2 telah berjaya dilakukan untuk menunjukkan tapak perlekatan faktor transkripsi AP4 berperanan sebagai unsur penindas dalam pengawalan pengekspresan ek2. Peningkatan aras AP4 telah dikaitkan dengan prognosis buruk karsinoma. Oleh itu, peranan pengawalaturan tapak perlekatan AP4 yang dilaporkan dalam kajian ini mungkin menghubungkaitkan ek2 dengan karsinogenesis. Walau pun kajian lanjut perlu dijalankan untuk memahami dan menentukan mekanisme penindasan AP4 dalam promoter ek2, namun pencirian dan analisis promoter ek2 yang dilakukan dalam kajian ini memberikan pengetahuan penting mengenai kawalan asas transkripsi ek2 yang dapat membantu pengawalan aras pengekspresan ek dalam keadaan patologi yang melibatkan gen ini.

 

Kata kunci: AP4; etanolamina kinase; faktor transkripsi; HepG2; promoter

REFERENCES

Bleijerveld, O.B., Brouwers, J.F., Vaandrager, A.B., Helms, J.B. & Houweling, M. 2007. The CDP-ethanolamine pathway and phosphatidylserine decarboxylation generate different phosphatidylethanolamine molecular species. Journal of Biological Chemistry 282: 28362-28372.

Chua, S.L., Khoo, B.Y., See Too, W.C. & Few, L.L. 2015. Expression profiling of choline and ethanolamine kinases in MCF7, HCT116 and HepG2 cells, and the transcriptional regulation by epigenetic modification. Molecular Medicine Reports 11: 611-618.

Cooper, S.J., Trinklein, N.D., Anton, E.D., Nguyen, L. & Myers, R.M. 2006. Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome. Genome Research 16: 1-10.

de Wet, J.R., Wood, K., DeLuca, M., Helinski, D.R. & Subramani, S. 1987. Firefly luciferase gene: Structure and expression in mammalian cells. Molecular and Cellular Biology 7: 725-737.

Egawa, T. & Littman, D.R. 2011. Transcription factor AP4 modulates reversible and epigenetic silencing of the Cd4 gene. Proceedings of the National Academy of Sciences 108: 14873-14878.

Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A.E., Kel, O.V., Ignatieva, E.V., Ananko, E.A., Podkolodnaya, O.A., Kolpakov, F.A., Podkolodny, N.L. & Kolchanov, N.A. 1998. Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Research 26: 362-367.

Hurley, T.M., McClive, P.J., Sarraj, M.A. & Sinclair, A.H. 2004. Eki2 is upregulated specifically in the testis during mouse sex determination. Gene Expression Patterns 4: 135-140.

Kent, W.J. 2002. BLAT--the BLAST-like alignment tool. Genome Research 12: 656-664.

Kim, M.Y., Jeong, B.C., Lee, J.H., Kee, H.J., Kook, H., Kim, N.S., Kim, Y.H., Kim, J.K., Ahn, K.Y. & Kim, K.K. 2006. A repressor complex, AP4 transcription factor and geminin, negatively regulates expression of target genes in nonneuronal cells. Proceedings of the National Academy of Sciences 103: 13074-13079.

Kiss, Z. 1999. Regulation of mitogenesis by water-soluble phospholipid intermediates. Cell Signaling 11: 149-57.

Kuan, C.S., See Too, W.C. & Few, L.L. 2016. Sp1 and Sp3 are the transcription activators of human ek1 promoter in TSA-treated human colon carcinoma cells. PLoS ONE 11: e0147886. doi:10.1371/journal.pone.0147886.

Liu, X., Bo, Z., Yan, G., Lei, W., Wu, C., Qi, L., Lin, Y., Tao, K., Wang, G. & Chen, J. 2012. The overexpression of AP-4 as a prognostic indicator for gastric carcinoma. Medical Oncology 29: 871-877.

Lykidis, A., Wang, J., Karim, M.A. & Jackowski, S. 2001. Overexpression of a mammalian ethanolamine-specific kinase accelerates the CDP-ethanolamine pathway. Journal of Biological Chemistry 276: 2174-2179.

Maston, G.A., Evans, S.K. & Green, M.R. 2006. Transcriptional regulatory elements in the human genome. Annual Review of Genomics and Human Genetics 7: 29-59.

Ow, D.W., de Wet, J.R., Helinski, D.R., Howell, S.H., Wood, K.V. & Deluca, M. 1986. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234: 856-859.

Quandt, K., Frech, K., Karas, H., Wingender, E. & Werner, T. 1995. MatInd and MatInspector: New fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Research 23: 4878-4884.

Sandelin, A., Carninci, P., Lenhard, B., Ponjavic, J., Hayashizaki, Y. & Hume, D.A. 2007. Mammalian RNA polymerase II core promoters: Insights from genome-wide studies. Nature Reviews Genetics 8: 424-436.

Shifera, A.S. & Hardin, J.A. 2010. Factors modulating expression of Renilla luciferase from control plasmids used in luciferase reporter gene assays. Analytical biochemistry 396: 167-172.

Smale, S.T. & Kadonaga, J.T. 2003. The RNA polymerase II core promoter. Annual Review of Biochemistry 72: 449-479.

Spanner, S. & Ansell, G.B. 1978. Choline kinase and ethanolamine kinase activity in the cytosol of nerve endings from rat forebrain. Biochemical Journal 178: 753-760.

Suzuki, Y., Taira, H., Tsunoda, T., Mizushima-Sugano, J., Sese, J., Hata, H., Ota, T., Isogai, T., Tanaka, T. & Morishita, S. 2001. Diverse transcriptional initiation revealed by fine, large-scale mapping of mRNA start sites. EMBO Reports 2: 388-393.

Thorne, N., Inglese, J. & Auld, D.S. 2010. Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chemistry & Biology 17: 646-657.

Tian, Y., Jackson, P., Gunter, C., Wang, J., Rock, C.O. & Jackowski, S. 2006. Placental thrombosis and spontaneous fetal death in mice deficient in ethanolamine kinase 2. Journal of Biological Chemistry 281: 28438-28449.

Vance, J.E. 2008. Thematic review series: Glycerolipids. phosphatidylserine and phosphatidylethanolamine in mammalian cells: Two metabolically related aminophospholipids. Journal of Lipid Research 49: 1377- 1387.

Walkey, C.J., Yu, L., Agellon, L.B. & Vance, D.E. 1998. Biochemical and evolutionary significance of phospholipid methylation. Journal of Biological Chemistry 273: 27043- 27046.

 

 

*Corresponding author; email: fewling@usm.my

 

 

 

 

 

 

previous