Transcriptional control analyses of the Xiphophorus melanoma oncogene.
Melanoma development in interspecific hybrids of Xiphophorus is induced by the overexpression of the mutationally activated receptor tyrosine kinase Xmrk in pigment cells. Based on the melanocyte specificity of the transcriptional upregulation, a pigment cell-specific promoter region was postulated for xmrk, the activity of which is controlled in healthy purebred fish by the molecularly still unidentified regulator locus R. However, as yet the xmrk promoter region is still poorly characterized. In order to contribute to a better understanding of xmrk expression regulation, we performed a functional analysis of the entire putative gene regulatory region of the oncogene using conventional plasmid-based reporter systems as well as a newly established method employing BAC-derived luciferase reporter constructs in melanoma and non-melanoma cell lines. Using the melanocyte-specific mitfa promoter as control, we could demonstrate that our in vitro system is able to reliably monitor regulation of transcription through cell type-specific regulatory sequences. We found that sequences within 200kb flanking the xmrk oncogene do not lead to any specific transcriptional activation in melanoma compared to control cells. Hence, xmrk reporter constructs fail to faithfully reproduce the endogenous transcriptional regulation of the oncogene. Our data therefore strongly indicate that the melanocyte-specific transcription of xmrk is not the consequence of pigment cell-specific cis-regulatory elements in the promoter region. This hints at additional regulatory mechanisms involved in transcriptional control of the oncogene, thereby suggesting a key role for epigenetic mechanisms in oncogenic xmrk overexpression and thereby in tumor development in Xiphophorus.
Physiological Chemistry, Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany.
Janine
J
Regneri
Institut de Génomique Fonctionelle de Lyon, Ecole Normale Supérieure de Lyon, 46, allée d'Italie, 69364 Lyon cedex 07, France.
Jean-Nicolas
JN
Volff
Physiological Chemistry, Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany; Comprehensive Cancer Center Mainfranken, University Clinic Würzburg, Würzburg, Germany. Electronic address: phch1@biozentrum.uni-wuerzburg.de.
Manfred
M
Schartl
D000818:Animals; D002460:Cell Line; D003532:Cyprinodontiformes / Q000235:genetics*; D029941:Fish Proteins / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D008545:Melanoma / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009857:Oncogenes / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics
D029941:Fish Proteins; D020794:Receptor Protein-Tyrosine Kinases; C077101:Xmrk protein, Xiphophorus
Cis-regulatory element; EGF receptor; Melanoma; Pigment cell; Transcriptional control; Tumor suppressor; Xiphophorus; xmrk oncogene