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사람 핵DNA로부터 FosB 유전자 프로모터 클로닝 및 활성도 분석

Cloning and Activity Analysis of the FosB Promoter Region from Human Genomic DNA

  • 나한흠 (강원대학교 자연과학대학 생명과학과) ;
  • 강윤성 (유디피아 분자진단 연구소) ;
  • 김근철 (강원대학교 자연과학대학 생명과학과)
  • Na, Han-Heom (Department of Biological Sciences, College of Natural Sciences, Kangwon National University) ;
  • Kang, Yoonsung (Institute for Diagnostic Markers, Eudipia Inc) ;
  • Kim, Keun-Cheol (Department of Biological Sciences, College of Natural Sciences, Kangwon National University)
  • 투고 : 2016.11.17
  • 심사 : 2017.03.13
  • 발행 : 2017.08.30

초록

FosB (FBJ murine osteosarcoma viral oncogene homolog B) 유전자는 사람의 19번 염색체에 위치하고 있으며 약 43 KD의 단백질을 코딩하며, 발생 및 분화과정, 개체 유지, 발병 진행 등을 조절한다고 알려져 왔다. 본 연구에서는 바이오 마커 등의 가능성이 있다고 보고된 FosB 유전자의 프로모터를 클로닝하여 활성도를 분석하고자 하였다. FosB genomic DNA 서열을 확인한 결과, TSS upstream 방향의 약 1 Kb 안쪽 부위에 FosB 유전자 발현을 위한 중요한 요소들이 있을 것으로 추정하였고, 따라서 FosB genomic DNA의 upstream -1,555 부위부터 exon 1의 +73까지 부위에 대한 PCR 증폭을 수행하였다. 또한 클로닝 성공을 높이기 위하여 일차로 $TA-1^{st}FosBp$ plasmid를 얻은 후, 다시 $TA-1^{st}FosBp$ plasmid를 template로 Kpn1과 Nhe1 제한 효소 절단부위를 프라이머에 삽입한 후 제작하여 2차 PCR을 수행하였으며, $TA-2^{nd}FosBp$ 플라스미드를 제작한 후 제한 효소로 절단하여 pGL3-luc vector로 subcloning하였다. 제작된 pGL3-FosBp-luc를 이용하여 항암제에 대한 활성도를 분석하고자 A549 사람 폐암세포주에 pGL3-FosBp-luc 플라스미드를 transfection 한 후 luciferase 활성도 분석을 수행하였다. Luciferase 활성도 증가는 doxorubicin, taxol 등을 처리한 후 단백질 발현 양상과 비교 하였을 때도 일치되는 결과를 얻을 수 있었다. 그러므로 FosB프로모터 클로닝은 향후 유전자 발현 연구, 마커분석 등에 유용할 것으로 사료된다.

The FBJ murine osteosarcoma viral oncogene homolog B (FosB) gene is located at chromosome 19, and encodes 43 Kda protein. Functionally, the FosB gene is important for differentiation, development, and pathogenesis. Furthermore, the FosB gene is suggested as possible biomarker for tracing disease prognosis. In this study, we constructed plasmid containing a FosB promoter region and evaluate its promoter activity. We analyzed the putative promoter region in FosB genomic DNA using bioinformatics program, and we found important regulatory elements in 1 Kb upstream from transcription start site (TSS). Therefore, we performed polymerase chain reaction (PCR) amplification on region from-1,555 upstream to +73 of the FosB genomic DNA, and PCR product was inserted into TA vector to create the $TA-1^{st}FosBp$ plasmid. We then prepared the primer sets, which contain a restriction enzyme site for Kpn1 and Nhe1, in order to reinsert into the TA vector to prepare $TA-2^{nd}FosBp$ plasmid. It was finally subcloned into pGL3-luc vector after enzyme cutting. To evaluate whether the cloned plasmid is useful in cell based experiment, we performed luciferase assay with pGL3-FosBp-luctransfection. FosB promoter activity was increased compared to empty vector, and this activity was significantly increased by treatment of doxorubicin and taxol. We obtained consistent data on regulation of FosB gene expression after anticancer drug treatment using Western blot analysis. The results suggest that promoter cloning of the human FosB gene is very useful for studying gene expression and analyzing biomarkers.

키워드

참고문헌

  1. Chandra, R., Francis, T. C., Konkalmatt, P., Amgalan, A., Gancarz, A. M., Dietz, D. M. and Lobo, M. K. 2015. Opposing role for Egr3 in nucleus accumbens cell subtypes in cocaine action. J. Neurosci. 35, 7927-7937. https://doi.org/10.1523/JNEUROSCI.0548-15.2015
  2. Chen, D. and Yang, H. 2015. Integrated analysis of differentially expressed genes in breast cancer pathogenesis. Oncol. Lett. 9, 2560-2566. https://doi.org/10.3892/ol.2015.3147
  3. Gajewski, P. A., Turecki, G. and Robison, A. J. 2016. Differential expression of FosB proteins and potential target genes in select brain regions of addiction and depression patients. PLoS One 11, e0160355. https://doi.org/10.1371/journal.pone.0160355
  4. Halder, K., Benzler, M. and Hartig, J. S. 2012. Reporter assays for studying quadruplex nucleic acids. Methods 57, 115-121. https://doi.org/10.1016/j.ymeth.2012.02.005
  5. Imbe, H. and Kimura, A. 2016. Repeated forced swim stress affects the expression of pCREB and DeltaFosB and the acetylation of histone H3 in the rostral ventromedial medulla and locus coeruleus. Brain Res. Bull. 127, 11-22. https://doi.org/10.1016/j.brainresbull.2016.08.007
  6. Jurado, J., Fuentes-Almagro, C. A., Prieto-Alamo, M. J. and Pueyo, C. 2007. Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species. BMC Mol. Biol. 8, 83. https://doi.org/10.1186/1471-2199-8-83
  7. Jurzak, M. and Adamczyk, K. 2013. Influence of genistein on c-Jun, c-Fos and Fos-B of AP-1 subunits expression in skin keratinocytes, fibroblasts and keloid fibroblasts cultured in vitro. Acta. Pol. Pharm. 70, 205-213.
  8. Knight, W. D., Little, J. T., Carreno, F. R., Toney, G. M., Mifflin, S. W. and Cunningham, J. T. 2011. Chronic intermittent hypoxia increases blood pressure and expression of FosB/DeltaFosB in central autonomic regions. Am. J. Physiol. Regul. Integr. Comp. Physiol. 301, R131-139. https://doi.org/10.1152/ajpregu.00830.2010
  9. Lazenka, M. F., David, B. G., Lichtman, A. H., Nestler, E. J., Selley, D. E. and Sim-Selley, L. J. 2014. Delta FosB and AP-1-mediated transcription modulate cannabinoid CB(1) receptor signaling and desensitization in striatal and limbic brain regions. Biochem. Pharmacol. 91, 380-389. https://doi.org/10.1016/j.bcp.2014.07.024
  10. Lobo, M. K., Zaman, S., Damez-Werno, D. M., Koo, J. W., Bagot, R. C., DiNieri, J. A., Nugent, A., Finkel, E., Chaudhury, D., Chandra, R., Riberio, E., Rabkin, J., Mouzon, E., Cachope, R., Cheer, J. F., Han, M. H., Dietz, D. M., Self, D. W., Hurd, Y. L., Vialou, V. and Nestler, E. J. 2013. DeltaFosB induction in striatal medium spiny neuron subtypes in response to chronic pharmacological, emotional, and optogenetic stimuli. J. Neurosci. 33, 18381-18395. https://doi.org/10.1523/JNEUROSCI.1875-13.2013
  11. Martin, T. A., Jayanthi, S., McCoy, M. T., Brannock, C., Ladenheim, B., Garrett, T., Lehrmann, E., Becker, K. G. and Cadet, J. L. 2012. Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens. PLoS One 7, e34236. https://doi.org/10.1371/journal.pone.0034236
  12. Martin-Gallardo, A., McCombie, W. R., Gocayne, J. D., FitzGerald, M. G., Wallace, S., Lee, B. M., Lamerdin, J., Trapp, S., Kelley, J. M. and Liu, L. I. 1992. Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q13.3. Nat. Genet. 1, 34-39. https://doi.org/10.1038/ng0492-34
  13. Milde-Langosch, K., Roder, H., Andritzky, B., Aslan, B., Hemminger, G., Brinkmann, A., Bamberger, C. M., Loning, T. and Bamberger, A. M. 2004. The role of the AP-1 transcription factors c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of mammary carcinomas. Breast Cancer Res. Treat. 86, 139-152. https://doi.org/10.1023/B:BREA.0000032982.49024.71
  14. Na, H. H., Noh, H. J., Cheong, H. M., Kang, Y. and Kim, K. C. 2016. SETDB1 mediated FosB expression increases the cell proliferation rate during anticancer drug therapy. BMB Rep. 49, 238-243. https://doi.org/10.5483/BMBRep.2016.49.4.031
  15. Nestler, E. J. 2015. FosB: a transcriptional regulator of stress and antidepressant responses. Eur. J. Pharmacol. 753, 66-72. https://doi.org/10.1016/j.ejphar.2014.10.034
  16. Park, S. J., Bae, H. S. and Park, J. C. 2015. Osteogenic differentiation and gene expression profile of human dental follicle cells induced by human dental pulp cells. J. Mol. Histol. 46, 93-106. https://doi.org/10.1007/s10735-014-9604-1
  17. Ting, C. H., Chen, Y. C., Wu, C. J. and Chen, J. Y. 2016. Targeting FOSB with a cationic antimicrobial peptide, TP4, for treatment of triple-negative breast cancer. Oncotarget 7, 40329-40347. https://doi.org/10.18632/oncotarget.9612
  18. Torres, O. V., McCoy, M. T., Ladenheim, B., Jayanthi, S., Brannock, C., Tulloch, I., Krasnova, I. N. and Cadet, J. L. 2015. CAMKII-conditional deletion of histone deacetylase 2 potentiates acute methamphetamine-induced expression of immediate early genes in the mouse nucleus accumbens. Sci. Rep. 5, 13396. https://doi.org/10.1038/srep13396
  19. Ulery, P. G., Rudenko, G. and Nestler, E. J. 2006. Regulation of DeltaFosB stability by phosphorylation. J. Neurosci. 26, 5131-5142. https://doi.org/10.1523/JNEUROSCI.4970-05.2006
  20. Vialou, V., Bagot, R. C., Cahill, M. E., Ferguson, D., Robison, A. J., Dietz, D. M., Fallon, B., Mazei-Robison, M., Ku, S. M., Harrigan, E., Winstanley, C. A., Joshi, T., Feng, J., Berton, O. and Nestler, E. J. 2014. Prefrontal cortical circuit for depression- and anxiety-related behaviors mediated by cholecystokinin: role of DeltaFosB. J. Neurosci. 34, 3878-3887. https://doi.org/10.1523/JNEUROSCI.1787-13.2014
  21. Vialou, V., Thibault, M., Kaska, S., Cooper, S., Gajewski, P., Eagle, A., Mazei-Robison, M., Nestler, E. J. and Robison, A. J. 2015. Differential induction of FosB isoforms throughout the brain by fluoxetine and chronic stress. Neuropharmacology 99, 28-37. https://doi.org/10.1016/j.neuropharm.2015.07.005
  22. Wang, H., Tao, X., Huang, S. T., Wu, L., Tang, H. L., Song, Y., Zhang, G. and Zhang, Y. M. 2016. Chronic Stress Is Associated with Pain Precipitation and Elevation in Delta Fosb Expression. Front. Pharmacol. 7, 138.
  23. Yoon, J., Kim, J. H., Lee, O. J., Lee, S. Y., Lee, S. H., Park, J. B., Lee, J. Y., Kim, S. C. and Kim, J. 2013. AP-1(c-Jun/FosB) mediates xFoxD5b expression in Xenopus early developmental neurogenesis. Int. J. Dev. Biol. 57, 865-872. https://doi.org/10.1387/ijdb.130163jk
  24. Zhang, H., Korenkova, V., Sjoback, R., Svec, D., Bjorkman, J., Kruhoffer, M., Verderio, P., Pizzamiglio, S., Ciniselli, C. M., Wyrich, R., Oelmueller, U., Kubista, M., Lindahl, T., Lonneborg, A. and Rian, E. 2014. Biomarkers for monitoring pre-analytical quality variation of mRNA in blood samples. PLoS One 9, e111644. https://doi.org/10.1371/journal.pone.0111644
  25. Zhang, Q., Liu, Q., Li, T., Liu, Y., Wang, L., Zhang, Z., Liu, H., Hu, M., Qiao, Y. and Niu, H. 2016. Expression and colocalization of NMDA receptor and FosB/DeltaFosB in sensitive brain regions in rats after chronic morphine exposure. Neurosci. Lett. 614, 70-76. https://doi.org/10.1016/j.neulet.2015.11.052