Mutagenesis Analysis of ABCG2 Gene Promoter of Zebrafish (Danio Rerio)
Abstract
Breast cancer is the commonest cancer among women worldwide and the probability of a woman dying from breast cancer is high (about 1 in 38 of total human population (2.6%)).The main factor for mortality is due to the resistance of this particular disease to chemotherapeutic agents. One of the most well-known proteins to be found to correlate significantly with breast cancer resistance to chemotherapeutic agent is the ATP-binding cassette super-family G member 2 (ABCG2). Knowledge on ABCG2 gene regulation is still lacking in terms of how the increased cytotoxic levels are closely related to induce a hype in gene transcript levels and ultimately cause of the reduction in chemotherapeutic agents. The approach taken in this study is through mutational analysis of selected transcription factor governing the expression of ABCG2. In order to achieve this, a previously cloned ABCG2 promoter which has been isolated (around 1500 bp in size) from Danio rerio and inserted into pGL3.0 plasmid, was subjected to site-directed mutagenesis. Selected transcription factor which is AP-1 was successfully mutated by deletion of 5'- TGACGCG -3' sequence at position 1113 bp from TSS+1 where it would bind in order to define their role in ABCG2 physiological function. Sequencing result after site-directed mutagenesis shows high similarities about 98% with ABCG2 gene of Danio rerio. Upon validation, it was found that the intended AP-1 binding site has been mutated. In future work, the mutated clone here will be subjected to transfection analysis where dual-luciferase assay will be conducted to verify the loss of activity from the ABCG2 promoter upon mutation of the targeted AP-1 site. Hence, the mutagenesis analysis of ABCG2 promoter are able to provide information on the involvement of AP-1 transcription factor in multidrug resistance mechanism of breast cancer and thus will be a potential target for chemotherapeutic agent.
References
Andersen, V., Vogel, L. K., Kopp, T. I., Sæbø, M., Nonboe, A. W., Hamfjord, J., Kure, E. H., & Vogel, U. (2015). High ABCC2 and Low ABCG2 Gene Expression Are Early Events in the Colorectal Adenoma-Carcinoma Sequence. Plos One, 10(3), 1-13.
https://doi.org/10.1371/journal.pone.0119255
Chung, H. H. (2018). Real-time polymerase chain reaction (RT-PCR) for the authentication of raw meats. International Food Research Journal, 25(2), 632-638.
Gazon, H., Barbeau, B., Mesnard, J.-M., & Peloponese, J.-M. (2018). Hijacking of the AP-1 Signaling Pathway during Development of ATL. Frontiers in Microbiology, 8, 2686.
https://doi.org/10.3389/fmicb.2017.02686
Grossman, S. R., Zhang, X., Wang, L., Engreitz, J., Melnikov, A., Rogov, P., Tewhey, R., Isakova, A., Deplancke, B., Bernstein, B. E., & Mikkelsen, T. S. (2017). Systematic dissection of genomic features determining transcription factor binding and enhancer function. Proceedings of the National Academy of Sciences, 114(7), E1291-E1300. doi:10.1073/pnas.1621150114
https://doi.org/10.1073/pnas.1621150114
Hernandez-Garcia, C. M., & Finer, J. J. (2014). Identification and validation of promoters and cis-acting regulatory elements. Plant Science, 217, 109-119.
https://doi.org/10.1016/j.plantsci.2013.12.007
Hu, J., Zhang, H., Liu, L., Han, B., Zhou, G., & Su, P. (2020). TRPS1 Confers Multidrug Resistance of Breast Cancer Cells by Regulating BCRP Expression. Frontiers in Oncology, 10,
https://doi.org/10.3389/fonc.2020.00934
doi:10.3389/fonc.2020.00934
https://doi.org/10.3389/fonc.2020.00934
Jee, M. S., Lim, L. W. K., Dirum, M. A., Hashim, S. I., Masri, M. S., Tan, H. Y., Lai, L. S., Yeo, F. K. S., &
Chung, H. H. (2017). Isolation and characterization of avirulence genes in Magnaporthe oryzae. Borneo Journal of Resource Science and Technology, 7(1), 31-42.
https://doi.org/10.33736/bjrst.389.2017
Karin, M., Liu, Z.-G., & Zandi, E. (1997). AP-1 function and regulation. Current Opinion in Cell Biology, 9(2), 240-246.
https://doi.org/10.1016/S0955-0674(97)80068-3
Kel, A. E., Gössling, E., Reuter, I., Cheremushkin, E., Kel-Margoulis, O. V., & Wingender, E. (2003). MATCH: A tool for searching transcription factor binding sites in DNA sequences. Nucleic Acids Research, 31(13), 3576-3579.
https://doi.org/10.1093/nar/gkg585
Kerppola, T. K., & Curran, T. (1993). Selective DNA bending by a variety of bZIP proteins. Molecular and Cell Biology, 13(9), 5479-5489.
https://doi.org/10.1128/MCB.13.9.5479
Kobayashi, I., Saito, K., Moritomo, T., Araki, K., Takizawa, F., & Nakanishi, T. (2008). Characterization and localization side population (SP) cells in zebrafish kidney hematopoietic tissue. Blood, 111(3), 1131-1137.
https://doi.org/10.1182/blood-2007-08-104299
Lim, L. W. K., & Chung, H. H. (in press). Functional characterization of ABCB4, ABCC1 and ACBG2 gene promoters in zebrafish (Danio rerio) embryos via microinjection reveal spatiotemporal xenobiotic multidrug resistance evidences. Gene Reports.
Lim, L. W. K., Chung, H. H., & Hussain, H. (2020). Organellar genome copy number variations and integrity across different organs, growth stages, phenotypes and main localities of sago palm (Metroxylon sagu Rottboll) in Sarawak. Gene Reports, 21, 100808. https://doi.org/10.1016/j.genrep.2020.100808.
https://doi.org/10.1016/j.genrep.2020.100808
Lim, L. W. K., Tan, H. Y., Aminan, A. W., Jumaan, A. Q., Moktar, M. Z., Tan, S. Y., Balinu, C. P., Robert, A. V., Chung, H. H. and Sulaiman, B. (2018a). Phylogenetic and expression of ATP-binding cassette
transporter genes in Rasbora sarawakenesis. Pertanika Journal of Tropical Agricultural Science, 41(3), 1341-1354.
Lim, L. W. K., Chung, H. H., Chong, Y. L., & Lee, N. K. (2018b). A survey of recently emerged genome-wide computational enhancer predictor tools. Computational Biology and Chemistry, 74(1), 132-141.
https://doi.org/10.1016/j.compbiolchem.2018.03.019
Lim, L. W. K., Chung, H. H., Chong, Y. L., & Lee, N. K. (2019a). Enhancers in proboscis monkey: A primer. Pertanika Journal of Tropical Agricultural Science, 42(1), 261-276.
Lim, L. W. K., Chung, H. H., Chong, Y. L., & Lee, N. K. (2019b). Isolation and characterization of putative liverspecific enhancers in proboscis monkey (Nasalis larvatus). Pertanika Journal of Tropical Agricultural Science, 42(2), 627-647.
Liu, R., & States, D. J. (2002). Consensus promoter identification in the human genome utilizing expressed gene markers and gene modeling. Genome Research, 12(3), 462-469.
https://doi.org/10.1101/gr.198002
Lu, C., Shen, Q., Dupré, E., Kim, H., Hilsenbeck, S., & Brown, P. H. (2005). cFos is critical for MCF-7 breast cancer cell growth. Oncogene, 24(43), 6516-6524.
https://doi.org/10.1038/sj.onc.1208905
Maciejczyk, A1., Szelachowska, J., Ekiert, M., Matkowski, R., Hałoń, A., & Surowiak, P. (2012). Analysis of BCRP expression in breast cancer patients. Ginekologia Polska, 83(9), 681-687.
Mishra, A., Dhanda, S., Siwach, P., Aggarwal, S., & Jayaram, B. (2020). A novel method SEProm for prokaryotic promoter prediction method based on DNA structure and energetics. Bioinformatics, 36(8), 2375-2384.
https://doi.org/10.1093/bioinformatics/btz941
Mo, W., & Zhang, J. T. (2012). Human ABCG2: structure, function, and its role in multidrug resistance. International Journal of Biochemistry and Molecular Biology, 3(1), 1-27.
Prusty, B. K., & Das, B. C. (2005). Constitutive activation of transcription factor AP-1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP-1 activity in HeLa cells by curcumin. International Journal of Cancer, 113(6), 951-960.
https://doi.org/10.1002/ijc.20668
Schinke, T., & Karsenty, G. (2008). Transcriptional Control of Osteoblast Differentiation and Function. Principles of Bone Biology, 1, 109-119.
https://doi.org/10.1016/B978-0-12-373884-4.00027-6
Shen, Q., Uray, I. P., Li, Y., Krisko, T. I., Strecker, T. E., Kim, H.-T., & Brown, P. H. (2007). The AP-1
transcription factor regulates breast cancer cell growth via cyclins and E2F factors. Oncogene, 27(3), 366-377.
https://doi.org/10.1038/sj.onc.1210643
Sukowati, C. H., Rosso, N., Pascut, D., Anfuso, B., Torre, G., Francalanci, P., Crocè, L. S., & Tiribelli, C. (2012). Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma. BMC
https://doi.org/10.1186/1471-230X-12-160
Gastroenterology, 12(1), 1-8.
Wang, S., Cheng, X., Li, Y., Wu, M., & Zhao, Y. (2018). Image-based promoter prediction: A promoter prediction method based on evolutionarily generated patterns. Scientific Reports, 8, 17695.
https://doi.org/10.1038/s41598-018-36308-0
Yip, C., Bhoo, PN., & Teo, S. (2014). A review of breast cancer research in Malaysia. Medical Journal of Malaysia, 69, 8-112
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