Mutagenesis Analysis of ABCB4 Gene Promoter of Danio rerio
Abstract
Zebrafish abcb4 gene (ortholog to human ABCB1 gene) serves primarily in multidrug resistance (MDR) mechanism by effluxing chemotherapeutic agents, chemicals, xenobiotics, and numerous anti-cancer drugs out of the cells. This study aims to identify the specific transcription factor binding sites (TFBS) within the promoter region of zebrafish abcb4 gene and determine the functional roles of these factors in abcb4 gene expression regulation via mutagenesis analysis. First, primers were designed to target and amplify the promoter region of zebrafish abcb4 gene through gradient PCR. The zebrafish abcb4 gene promoter was then cloned into pGL3.0 vector and sent for sequencing. The sequencing results revealed high similarity to zebrafish DNA sequence from clone DKEY-24I24 in linkage group 16, indicating a successful cloning of targeted gene. Thereafter, consensus sequence of zebrafish abcb4 gene promoter was generated with the length of 1,392 bp which was close to its expected size during primer design (1,500 bp). Using MATCH tool, 155 TFBSs were found within zebrafish abcb4 gene promoter region. Activator protein 1 (AP-1) TFBS at 1,255 bp was chosen to be mutated through site-directed mutagenesis. Mutagenic primers (forward primer: 5’ GGG CAA GGC AGT ATA AAC GTG 3’ and reverse primer: 5’ TTA TGT TTC TAG GGA TTA CGT CAC 3’) were designed to substitute AGT with GGG to remove the AP-1 TFBS. By mutating the zebrafish abcb4 gene promoter, the MDR phenomenon driven by zebrafish abcb4 gene can be elucidated and this might provide clues to the development of tumor and malignancy in human. The results from this study may enrich the knowledge in chemotherapy and cancer treatments.
References
Abanda, N. N., Riches, Z., & Collier, A. C. (2017). Lobular distribution and variability in hepatic ATP binding cassette protein B1 (ABCB1, P-gp): Ontogenetic differences and potential for toxicity. Pharmaceutics, 9(1),8.
https://doi.org/10.3390/pharmaceutics9010008
Annilo, T., Chen, Z., Shulenin, S., Costantino, J., Thomas, L., Lou, H., Stefanov, S., & Dean, M. (2006). Evolution of the vertebrate ABC gene family: Analysis of gene birth and death. Genomics, 88(1), 1-11.
https://doi.org/10.1016/j.ygeno.2006.03.001
Azizah, A. M., Hashimah, B., Nirmal, K., Siti Zubaidah, A. R., Puteri, N. A., Nabihah, A., Sukumaran, R., Balqis, B., Nadia, S. M. R., Sharifah, S. S. S., Rahayu, O., Nur Alham, O., & Azlina, A. A. (2019). Malaysia National Cancer Registry Report 2012-2016. Putrajaya, Malaysia: National Cancer Registry.
Chauhan, T. (2019). Site-directed mutagenesis: Role and applications. Retrieved from https://geneticeducation.co.in/ site-directed-mutagenesis -methods-and- applications/
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.
Daschner, P. J., Ciolino, H. P., Plouzek, C. A., & Yeh, G. C. (1999). Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells. Breast Cancer Research and Treatment, 53(3), 229-240.
https://doi.org/10.1023/A:1006138803392
Dermauw, W., & Van Leeuwen, T. (2014). The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance. Insect Biochemistry and Molecular Biology, 45, 89-110.
https://doi.org/10.1016/j.ibmb.2013.11.001
Ferreira, M., Costa, J., & Reis-Henriques, M. A. (2014). ABC transporters in fish species: A review. Frontiers in Physiology, 5, 266.
https://doi.org/10.3389/fphys.2014.00266
Fischer, S., Klüver, N., Burkhardt-Medicke, K., Pietsch, M., Schmidt, A., Wellner, P., Schirmer, K., &
Luckenback, T. (2013). Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos. BMC Biology, 11(69).
https://doi.org/10.1186/1741-7007-11-69
Foppoli, C., Coccia, R., & Perluigi, M. (2014). Role of oxidative stress in human papillomavirus-driven cervical carcinogenesis. In Cancer 51-61. Academic Press.
https://doi.org/10.1016/B978-0-12-405205-5.00006-4
Gustems, M., Woellmer, A., Rothbauer, U., Eck, S. H., Wieland, T., Lutter, D., & Hammerschmidt, W. (2014). c-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs. Nucleic Acids Research, 42(5), 3059-3072.
https://doi.org/10.1093/nar/gkt1323
Hamli, A. (2019). Functional characterization of plasmid harbouring ABCB4 gene promoter in zebrafish embryos. (Unpublished degree's thesis). University of Malaysia Sarawak, Kuching, Malaysia.
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
Hsieh, P. C., & Vaisvila, R. (2013). Protein engineering: Single or multiple site-directed mutagenesis. Methods in Molecular Biology, 173-186.
https://doi.org/10.1007/978-1-62703-293-3_13
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
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
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. Pertanika Journal of Tropical Agricultural Science.
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
Liu, X., Li, S., Peng, W., Feng, S., Feng, J., Mahboob, S., Al-Ghanim, K. A., & Xu, P. (2016). Genome-wide identification, characterization and phylogenetic analysis of ATP-binding cassette (ABC) transporter genes in common carp (Cyprinus carpio). PLoS One, 11(4).
https://doi.org/10.1371/journal.pone.0153246
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
Stavrovskaya, A. A. (2000). Cellular mechanisms of multidrug resistance of tumor cells. Biochemistry (Moscow), 65(1), 95-106.
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
Yu, D., Guo, S., Jing, Y., Dong, Y., & Wei, L. (2015). A review on hepatocyte nuclear factor-1beta and tumor.Cell & Bioscience, 5(1), 58.
https://doi.org/10.1186/s13578-015-0049-3
Zebrafish Information Network. (2019). Retrieved from https://zfin.org/ZDB-GENE-080204-52#
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