BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Functional characterization of a minimal sequence essential for the expression of human TLX2 gene

  • Borghini, Silvia (Laboratorio di Genetica Molecolare, Istituto G Gaslini, Universita di Genova) ;
  • Bachetti, Tiziana (Laboratorio di Genetica Laboratorio di Genetica Molecolare, Istituto G Gaslini, Universita di Genova) ;
  • Fava, Monica (Laboratorio di Genetica Molecolare, Istituto G Gaslini, Universita di Genova) ;
  • Duca, Marco Di (Laboratorio di Fisiopatologia dell'Uremia Universita di Genova) ;
  • Ravazzolo, Roberto (Laboratorio di Genetica Molecolare, Istituto G Gaslini, Universita di Genova) ;
  • Ceccherini, Isabella (Laboratorio di Genetica Molecolare, Istituto G Gaslini, Universita di Genova)
  • Published : 2009.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

TLX2 is an orphan homeodomain transcription factor whose expression is mainly associated with tissues derived from neural crest cells. Recently, we have demonstrated that PHOX2A and PHOX2B are able to enhance the neural cell-type specific expression of human TLX2 by binding distally the 5' -flanking region. In the present work, to deepen into the TLX2 transcription regulation, we have focused on the proximal 5'-flanking region of the gene, mapping the transcription start site and identifying a minimal promoter necessary and sufficient for the basal transcription in cell lines from different origin. Site-directed mutagenesis has allowed to demonstrate that the integrity of this sequence is crucial for gene expression, while electrophoretic mobility shift assays and chromatin immunoprecipitation experiments have revealed that such an activity is dependent on the binding of a PBX factor. Consistent with these findings, such a basal promoter activity has resulted to be enhanced by the previously reported PHOX2-responding sequence.

Keywords

References

  1. Roberts, C., Sonder, A., Lumsden, A. and Korsmeyer, S. (1995) Development expression of Hox11 and specification of splenic cell fate. Am. J. Pathol. 146, 1089-1101
  2. Hatano, M., Iitsuka, Y., Yamamoto, H., Dezawa, M., Yusa, S., Kohno, Y. and Tokuhisa, T. (1997) Ncx, a Hox11 related gene, is expressed in a variety of tissues derived from neural crest cells. Anat. Embryol. (Berl) 195, 419-425 https://doi.org/10.1007/s004290050061
  3. Shirasawa, S., Yunker, A., Roth, K., Brown, G., Horning, S. and Korsmeyer, S. (1997) Enx (Hox11L1)-deficient mice develop myenteric neuronal hyperplasia and megacolon. Nat. Med. 3, 646-650 https://doi.org/10.1038/nm0697-646
  4. Dear, T., Sanchez-Garcia, I. and Rabbitts, T. (1993) The HOX11 gene encodes a DNA-binding nuclear transcription factor belonging to a distinct family of homeobox genes. Proc. Natl. Acad. Sci. U.S.A. 90, 4431-4435 https://doi.org/10.1073/pnas.90.10.4431
  5. Shirasawa, S., Arata, A., Onimaru, H., Roth, K., Brown, G., Horning, S., Arata, S., Okumura, K., Sasazuki, T. and Korsmeyer, S. (2000) Rnx deficiency results in congenital central hypoventilation. Nat. Genet. 24, 287-290 https://doi.org/10.1038/73516
  6. Brake, R., Kees, U. and Watt, P. (1998) Multiple negative elements contribute to repression of the HOX11 proto-oncogene. Oncogene 17, 1787-1795 https://doi.org/10.1038/sj.onc.1202078
  7. Li, Y., Jiang, Z., Chen, H., Leung, W., Sung, J. and Ma, W. (2005) The existence of a putative regulatory element in 3'-untranslated region of proto-oncogene HOX11's mRNA. J. Biochem. Mol. Biol. 38, 500-506 https://doi.org/10.5483/BMBRep.2005.38.4.500
  8. Borghini, S., Vargiolu, M., Di Duca, M., Ravazzolo, R. and Ceccherini, I. (2006) Nuclear factor Y drives basal transcription of the human TLX3, a gene overexpressed in T-cell acute lymphocytic leukemia. Mol. Cancer. Res. 4, 635-643 https://doi.org/10.1158/1541-7786.MCR-05-0250
  9. Borghini, S., Bachetti, T., Fava, M., Di Duca, M., Cargnin, F., Fornasari, D., Ravazzolo, R. and Ceccherini, I. (2006) The TLX2 homeobox gene is a transcriptional target of PHOX2B in neural-crest-derived cells. Biochem. J. 395, 355-361 https://doi.org/10.1042/BJ20051386
  10. Borghini, S., Di Duca, M., Santamaria, G., Vargiolu, M., Bachetti, T., Cargnin, F., Pini Prato, A., De Giorgio, R., Lerone, M., Stanghellini, V., Jasonni, V., Fornasari, D., Ravazzolo, R. and Ceccherini, I. (2007) Transcriptional regulation of TLX2 and impaired intestinal innervation: possible role of the PHOX2A and PHOX2B genes. Eur. J. Hum. Genet. 15, 848-855 https://doi.org/10.1038/sj.ejhg.5201852
  11. Tang, S., Hoodless, P., Lu, Z., Breitman, M., McInnes, R., Wrana, J. and Buchwald, M. (1998) The Tlx-2 homeobox gene is a downstream target of BMP signalling and is required for mouse mesoderm development. Development 125, 1877-1887
  12. Hatano, M., Aoki, T., Dezawa, M., Yusa, S., Iitsuka, Y., Koseki, H., Taniguchi, M. and Tokuhisa, T. (1997) A novel pathogenesis of megacolon in Ncx/Hox11L.1 deficient mice. J. Clin. Invest. 100, 795-801 https://doi.org/10.1172/JCI119593
  13. Parisi, M., Baldessari, A., Iida, M., Clarke, C., Doggett, B., Shirasawa, S. and Kapur, R. (2003) Genetic background modifies intestinal pseudo-obstruction and the expression of a reporter gene in Hox11L1-/- mice. Gastroenterology 125, 1428-1440 https://doi.org/10.1016/j.gastro.2003.08.021
  14. Brake, R., Kees, U. and Watt, P. (2002) A complex containing PBX2 contributes to activation of the proto-oncogene HOX11. Biochem. Biophys. Res. Commun. 294, 23-34 https://doi.org/10.1016/S0006-291X(02)00426-6
  15. Lu, Q., Wright, D. and Kamps, M. (1994) Fusion with E2A converts the Pbx1 homeodomain protein into a constitutive transcriptional activator in human leukemias carrying the t(1;19) translocation. Mol. Cell. Biol. 14, 3938-3948 https://doi.org/10.1128/MCB.14.6.3938
  16. Iitsuka, Y., Shimizu, H., Kang, M., Sasagawa, K., Sekiya, S., Tokuhisa, T. and Hatano, M. (1999) An enhancer element for expression of the Ncx (Enx, Hox11L1) gene in neural crest-derived cells. J. Biol. Chem. 274, 24401-24407 https://doi.org/10.1074/jbc.274.34.24401
  17. Mann, R. and Affolter, M. (1998) Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8, 423-429 https://doi.org/10.1016/S0959-437X(98)80113-5
  18. Moens, C. and Selleri, L. (2006) Hox cofactors in vertebrate development. Dev. Biol. 291, 193-206 https://doi.org/10.1016/j.ydbio.2005.10.032
  19. Laurent, A., Bihan, R., Omilli, F., Deschamps, S. and Pellerin, I. (2008) PBX proteins: much more than Hox cofactors. Int. J. Dev. Biol. 52, 9-20 https://doi.org/10.1387/ijdb.072304al
  20. Wagner, K., Mincheva, A., Korn, B., Lichter, P. and P$\ddot{o}$pperl, H. (2001) Pbx4, a new Pbx family member on mouse chromosome 8, is expressed during spermatogenesis. Mech. Dev. 103, 127-131 https://doi.org/10.1016/S0925-4773(01)00349-5
  21. Di Giacomo, G., Koss, M., Capellini, T., Brendolan, A., P$\ddot{o}$pperl, H. and Selleri, L. (2006) Spatio-temporal expression of Pbx3 during mouse organogenesis. Gene Expr. Patterns 6, 747-757 https://doi.org/10.1016/j.modgep.2005.12.002
  22. Asahara, H., Dutta, S., Kao, H., Evans, R. and Montminy, M. (1999) Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner. Mol. Cell. Biol. 19, 8219-8225 https://doi.org/10.1128/MCB.19.12.8219
  23. Brendolan, A., Ferretti, E., Salsi, V., Moses, K., Quaggin, S., Blasi, F., Cleary, M. and Selleri, L. (2005) A Pbx1-dependent genetic and transcriptional network regulates spleen ontogeny. Development 132, 3113-3126 https://doi.org/10.1242/dev.01884
  24. Berkes, C., Bergstrom, D., Penn, B., Seaver, K., Knoepfler, P. and Tapscott, S. (2004) Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential. Mol. Cell 14, 465-477 https://doi.org/10.1016/S1097-2765(04)00260-6
  25. Fava, M., Borghini, S., Cinti, R., Cusano, R., Seri, M., Lerone, M., De Giorgio, R., Stanghellini, V., Martucciello, G., Ravazzolo, R. and Ceccherini, I. (2002) HOX11L1: a promoter study to evaluate possible expression defects in intestinal motility disorders. Int. J. Mol. Med. 10, 101-106
  26. Higuchi, R., Krummel, B. and Saiki, R. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic. Acids. Res. 16, 7351-7367 https://doi.org/10.1093/nar/16.15.7351
  27. Dignam, J., Lebovitz, R. and Roeder, R. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic. Acids. Res. 11, 1475-1489 https://doi.org/10.1093/nar/11.5.1475