Transcriptional regulation of cancer genes in the Xiphophorus melanoma system Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Janine Regneri aus Herdecke Würzburg 2013 Eingereicht am: ....................................................................................................................... Mitglieder der Promotionskommission: Vorsitzender: .......................................................................................................................... Gutachter : .............................................................................................................................. Gutachter: ............................................................................................................................... Tag des Promotionskolloquiums: ............................................................................................. Doktorurkunde ausgehändigt am: ............................................................................................ Eidesstattliche Erklärung Gemäß §4, Abs. 3, Ziff. 3, 5 und 8 der Promotionsordnung der Fakultät für Biologie der Bayerischen Julius-Maximilians-Universität Würzburg Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbständig angefertigt und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. Ich erkläre weiterhin, dass die vorliegende Dissertation weder in gleicher, noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat. Weiterhin erkläre ich, dass ich außer den mit dem Zulassungsantrag urkundlich vorgelegten Graden keine weiteren akademischen Grade erworben oder zu erwerben versucht habe. Würzburg, Juni 2013 ________________________________________ Janine Regneri Table of contents Abstract ...................................................................................................................................... 1 Zusammenfassung ...................................................................................................................... 3 Introduction ................................................................................................................................ 5 The Xiphophorus melanoma model ........................................................................................ 5 The xmrk oncogene ................................................................................................................. 7 Activating mutations in the Xmrk receptor......................................................................... 7 Oncogenic signaling of Xmrk ............................................................................................. 8 Control of xmrk expression ................................................................................................. 9 Genomic organization of the Tu locus .................................................................................. 11 Different mdl-xmrk allele combinations ........................................................................... 12 The R locus ........................................................................................................................... 14 Aim of the thesis ................................................................................................................... 16 Materials and methods ............................................................................................................. 17 Cell culture ........................................................................................................................... 17 Experimental animals ........................................................................................................... 17 RNA isolation, reverse transcription and expression analysis.............................................. 19 Protein isolation and Western blot analysis .......................................................................... 21 Generation of BAC reporter constructs ................................................................................ 22 Luciferase assay .................................................................................................................... 25 Preparation of gDNA, PCR and sequencing ........................................................................ 25 Phylogenetic analysis ........................................................................................................... 27 Comparative analysis of genomic sequences ....................................................................... 27 Results ...................................................................................................................................... 28 Analysis of the transcriptional regulation of different mdl-xmrk allele combinations ......... 28 Expression of the proto-oncogene egfrb in adult tissues of X. maculatus and X. hellerii 28 Expression of the tumorigenic mdl Sd -xmrk B allele ............................................................ 30 Expression of the proto-oncogene egfrb in xmrk B-expressing tissues of the black pigment cell lineage ........................................................................................................................ 35 Expression of the non-tumorigenic mdl Sr -xmrk A allele ..................................................... 36 Expression of Sr’’ and DrLi (mut) mutant alleles ............................................................ 39 Comparison of mdl Sr -xmrk A and mdl Sd -xmrk B expression in gills of X. maculatus and backcross hybrids .............................................................................................................. 41 Structural analysis of the proximal 5’ region of different mdl-xmrk alleles ..................... 42 Comparative structural analysis of the mdl Sr -xmrk A and mdl Sd -xmrk B genomic regions .. 43 Transcriptional activity of mdl Sr -xmrk A and mdl Sd -xmrk B reporter constructs .................. 48 Analysis of a deletion series of the mdl Sd -xmrk B reporter construct ................................. 51 Analysis of candidates for the R locus-encoded gene .......................................................... 54 mtap ................................................................................................................................... 55 pdcd4a ............................................................................................................................... 57 cxxc4 ................................................................................................................................. 58 cdkn2ab ............................................................................................................................. 59 Discussion ................................................................................................................................ 63 Transcriptional regulation of mdl-xmrk allele combinations ................................................ 63 Functional and structural characterization of the putative xmrk promoter region ................ 70 Analysis of R locus candidate genes ..................................................................................... 75 Perspectives .......................................................................................................................... 80 Appendix .................................................................................................................................. 81 Bibliography ............................................................................................................................. 94 Acknowledgments .................................................................................................................. 104 Abstract 1 Abstract The Xiphophorus melanoma system is a useful animal model for the study of the genetic basis of tumor formation. The development of hereditary melanomas in interspecific hybrids of Xiphophorus is connected to pigment cell specific overexpression of the mutationally activated receptor tyrosine kinase Xmrk. In purebred fish the oncogenic function of xmrk is suppressed by the molecularly still unidentified locus R. The xmrk oncogene was generated by a gene duplication event from the Xiphophorus egfrb gene and thereby has acquired a new 5’ regulatory sequence, which has probably altered the transcriptional control of the oncogene. So far, the xmrk promoter region was still poorly characterized and the molecular mechanism by which R controls xmrk -induced melanoma formation in Xiphophorus still remained to be elucidated. To test the hypothesis that R controls melanoma development in Xiphophorus on the transcriptional level, the first aim of the thesis was to gain a deeper insight into the transcriptional regulation of the xmrk oncogene. To this end, a quantitative analysis of xmrk transcript levels in different Xiphophorus genotypes carrying either the highly tumorigenic xmrk B or the non-tumorigenic xmrk A allele was performed. I was able to demonstrate that expression of the tumorigenic xmrk B allele is strongly increased in malignant melanomas of R-free backcross hybrids compared to benign lesions, macromelanophore spots, and healthy skin. The expression level of the non-tumorigenic xmrk A allele, in contrast, is not influenced by the presence or absence of R. These findings strongly indicate that differential transcriptional regulation of