Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Transcriptional Regulation of the Murine Dopamine Receptor Regulating Factor (DRRF) Gene

생쥐 도파민 수용쳬 조절인자 (DRRF) 유전자의 전사조절

  • Kim Ok Soo (Department of Bioscience and Biotechnology, Silla University) ;
  • Lee Young-Choon (Division of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University) ;
  • Lee Sang-Hyeon (Department of Bioscience and Biotechnology, Silla University)
  • Published : 2005.02.01
Download PDF
⟨ Previous Next ⟩

Abstract

The murine dopamine receptor regulating factor (DRRF) gene is transcribed from a TATA-less promoter that has several putative Sp1 binding sites. The present investigation identifies functional transcription factors that modulate the expression of this gene, In the $D_2-expressing$ NB41A3 cells, Spl potently activates transcription from the DRRF promoter in pCAT-DRRF-1153/+17, but DRRF effectively inhibits it. Deletion of the 31 bp fragment between -1153 and -1122 decreased transcription down to about $60\%$. This fragment contains a functional API binding site. In addition, deletion of the 129 bp region between -901 and -772 further decreased transcription. The latter region has a functional AP2 binding site. Using a DRRF_AP1 (bases -1153 to -1121) probe, a specific retarded band was observed, and the unlabeled AP1 consensus competitor could effectively compete away this retarded band. In addition, using a DRRF_AP2 (bases -873 to -846), a specific retarded band was observed, and the unlabeled AP2 consensus competitor could effectively compete away this retarded band. The present observations suggest that Spl and DRRF regulate the DRRF promoter and that both API and AP2 also modulate this gene.

생쥐의 도파민 수용체 조절인자 (DRRF)유전자는 몇몇 Spl 결합부위를 가지고 TATA가 없는 프로모터로부터 전사된다. 본 연구에서는 이 유전자의 발현을 조절하는 기능성 조절인자들을 밝힌다. $D_2$ 도파민 수용체를 발현하는 NB41A3 세포에서 Spl은 pCAT-DRRF-l153/+17에 포함된 DRRF 프로모터부터 의 전사를 촉진시키지만 DRRF는 전사를 억제시켰다. -1153과 -1122 사이의 31 bp 단편의 결손에 의해 전사활성은 약 $60\%$ 정도 감소하였다. 이 단편은 기능성 AP1 결합부위를 포함하고 있다 게다가, -901과 -772사이의 129 bp영역의 결손에 의해 전사활성이 더욱 더 감소하였다. 이 영역은 기능성 AP2 결합부위를 가진다. DRRF_AP1 (bases -1153 to -1121) 탐침을 이용한 gel shift실험에서 특정 벤드가 관찰되었고, 이 벤드는 API 상보성 경쟁자에 의해 효과적으로 사라졌다. 더욱이, DRRF_AP2 (bases -873 to -846) 탐침을 이용한 gel shift실험에서도 특정 벤드가 관찰되었고, 이 벤드도 AP2상보성 경쟁자에 의해 효과적으로 사라졌다. 본 연구결과로, Spl과 DRRF가 DRRF 프로모터를 효과적으로 조절한다는 사실과, AP1과 AP2 역시 이 유전자를 조절한다는 사실을 알 수 있었다.

Keywords

References

  1. Ausubel, F. M., R. Brent, R. E., Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Strohl. 1989. Current protocols in molecular biology, pp. 12.2.1-12.2.10, John Wiley and Sons, Inc., New York
  2. Dignam, J. D., R. M. Lebovitz and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Res. 11, 1475-1489 https://doi.org/10.1093/nar/11.5.1475
  3. Harrison, S. C. 1991. A structural taxotomy of DNA-binding domains. Nature 353, 715-719 https://doi.org/10.1038/353715a0
  4. Hennighausen, L. and H. Lubon. 1987. Interaction of protein with DNA in vitro. Methods Enzymol. 152, 721-735 https://doi.org/10.1016/0076-6879(87)52076-6
  5. Hwang, C. K., U. M. D'Souza, A. J. Eisch, S. Yajima, C.-H. Lammers, Y. Yang, S.-H. Lee, Y.-M. Kim, E. J. Nestler and M. M. Mouradian. 2001. Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor. Proc. Natl. Acad. Sci. USA 98, 7558-7563 https://doi.org/10.1073/pnas.121635798
  6. Imhof, A., M. Schuierer, O. Werner, M. Moser, C. Roth, R. Bauer and R. Buettner. 1999. Transcriptional regulation of the AP-2${\alpha}$ promoter by BTEB-1 and AP- 2rep, a novel wt-1/egr-related zinc finger repressor. Mol. Cell. Biol. 19, 194-204
  7. Imataka, H., K. Sogawa, K.-I. Yasumoto, Y. Kikuchi, K. Sasano, A. Kobayashi, M. Hayami and Y. Fujii- Kurayama. 1992. Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P501A1 gene. EMBO J. 11, 3663-3671
  8. Kaczynski, J. A., A. A. Conley, M. Fernandez Zapico, S. M. Delgado, J.-S. Zhang and R. Urrutia. 2002. Functional analysis of basic transcription element (BTE)-binding protein (BTEB) 3 and BTEB4, a novel Spl-like protein, reveals a subfamily of transcriptional repressors for the BTE site of the cytochrome P4501AI gene promoter. Biochem. J. 366, 873-882
  9. Lee, S.-H., Y.-M. Kim, S. Yajima, J.-M. Ha, B. J. Ha, O. S. Kim, K. Ohsawa and M. M. Mouradian. 2003. Genomic organization and promoter characterization of the murine dopamine receptor regulating factor (DRRF) gene. Gene 304, 193-199 https://doi.org/10.1016/S0378-1119(02)01209-X
  10. Martin, K. M., W. N. Cooper, J. C. Metcalfe and P. R. Kemp. 2000. Mouse BTEB3, a new member of the basic transcription element binding protein (BTEB) family, activates expression from GC-rich minimal promoter regions. Biochem. J. 345, 529-533 https://doi.org/10.1042/0264-6021:3450529
  11. Minowa, T., M. T. Minowa and M. M. Mouradian. 1994. Negative modulator of the rat D2 dopamine receptor gene. J. Biol. Chem. 269, 11656-11662
  12. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  13. Simmen, R. C. M., T. E. Chung, H. Imataka, F. J. Michel, L. Badinga and F. A. Simmen. 1999. Trans-activation functions of the Sp-related nuclear factor, basic transcription element-binding protein, and progesterone receptor in endometrial epithelical cells. Endocrinol. 140, 2517-2525 https://doi.org/10.1210/en.140.6.2517
  14. Yajima, S., S.-H. Lee, T. Minowa, and M. M. Mouradian. 1998. Sp family transcription factors regulates expression of rat $D_2$ dopamine receptor gene. DNA & Cell Biol. 17, 471-479 https://doi.org/10.1089/dna.1998.17.471