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Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential Iigand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properlies and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related Iigand-binding domain sequences of the human epidennal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk proteinwas examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transfonning protein equivalent to other tyrosine kinase oncoproteins.
Das maligne Melanom ist ein Hauttumor mit steigender Inzidenz und hohen Mortalitätsraten. Da die molekularbiologischen Ereignisse, die der Melanomentwicklung zugrundeliegen, nur unzureichend bekannt sind, gibt es kaum spezifische Therapieansätze. Zur Untersuchung der Melanomentwicklung eignet sich das Xiphophorus-Modell. In diesem System ist die Anwesenheit der RTK Xmrk ausreichend, um durch Aktivierung proliferativer und entdifferenzierender Signalwege und Apoptoseinhibition Melanome zu verursachen. Im Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass Xmrk auch die Migration der Melanozytenzellinie Melan a-Hm induzieren kann. Die Migration der durch Xmrk transformierten Zellen ist amöboid und unabhängig von MAPK- und PI3K-Signalwegen. Eine Funktion bei der Migration haben jedoch die Kinasen FAK und Fyn. Sie bilden möglicherweise einen Proteinkomplex, der für FAK und Src aus zahlreichen anderen Systemen bekannt ist und als Signalplattform für die Zellmigration fungiert. Diese Erkenntnisse können dazu beitragen, das Xiphophorus-Modell weiterzuentwickeln und die Grundlagen der Melanomgenese besser zu verstehen.
Melanome stellen die gefährlichste Form von Hautkrebs mit der höchsten Mortalitätsrate dar. Der Transformation normaler Melanozyten zu malignen Melanomen liegen komplexe molekulare und biochemische Veränderungen zu Grunde. Im Xiphophorus-Melanom-Modell ist die onkogene Rezeptortyrosinkinase "Xiphophorus melanoma receptor kinase" (Xmrk) der alleinige Auslöser der Melanominitiation und -progression. Die Aufklärung der Xmrk-vermittelten Signaltransduktion kann zum besseren Verständnis von Ereignissen, die auch bei der humanen Melanomentwicklung eine Rolle spielen, beitragen. In der vorliegenden Arbeit wurde mit Hilfe der Microarray-Technologie die Regulation der Genexpression durch Xmrk analysiert. Zu den nach Rezeptoraktivierung am stärksten herabregulierten Genen gehörten "son of sevenless homolog 1" (Sos1) und "ubiquitin-conjugating enzyme E2I" (Ube2i); stark hochreguliert waren "early growth response 1" (Egr1), "cysteine-rich protein 61" (Cyr61), "dual-specificity phosphatase 4" (Dusp4), "fos-like antigen 1" (Fosl1), "epithelial membrane protein" (Emp1), Osteopontin (Opn), "insulin-like growth factor binding protein 3" (Igfbp3) und "tumor-associated antigen L6" (Taal6). Die für die Regulation dieser Gene verantwortlichen Signalwege wurden durch die Anwendung von niedermolekularen Inhibitoren und siRNA identifiziert, wobei für die SRC-Kinase FYN eine zentrale Bedeutung bei der Xmrk-abhängigen Regulation der Genexpression festgestellt wurde. Darüber hinaus wurde die Expression der Gene in humanen Melanomzelllinien im Vergleich zu normalen humanen Melanozyten untersucht. Als besonders vielversprechende Kandidaten stellten sich dabei DUSP4 und TAAL6 heraus, deren Rolle in der humanen Melanominduktion und -progression Gegenstand zukünftiger Studien sein wird. In einem anderen Ansatz zur Aufklärung des Signalnetzwerkes sollten Zielproteine von Xmrk durch Protein-Protein-Interaktionsstudien mit Hilfe des Split-Ubiquitin-Systems ermittelt werden. Aufgrund ungünstiger Expressions- oder Faltungseigenschaften von Xmrk in diesem System war es aber nicht möglich, den Rezeptor als Köderprotein einzusetzen. Das für die Xmrk-vermittelte Melanomentstehung zentrale Protein FYN konnte jedoch als Köder etabliert und seine Wechselwirkung mit der Tyrosinkinase FAK analysiert werden. Es wurde gezeigt, dass der phosphorylierte Tyrosinrest an Position 397 von FAK für die Interaktion einer N-terminal trunkierten FAK-Variante mit FYN notwendig ist und dass diese Phosphorylierung in Hefe gewährleistet zu sein scheint. Die Suche nach neuen Interaktionspartnern von FYN mittels der Split-Ubiquitin-Technologie könnte Einblicke in weitere FYN-abhängige Ereignisse bieten, die zur Aufklärung seiner zentralen Rolle bei der Tumorentstehung dienen könnte.
Background
Xiphophorus fishes are represented by 26 live-bearing species of tropical fish that express many attributes (e.g., viviparity, genetic and phenotypic variation, ecological adaptation, varied sexual developmental mechanisms, ability to produce fertile interspecies hybrids) that have made attractive research models for over 85 years. Use of various interspecies hybrids to investigate the genetics underlying spontaneous and induced tumorigenesis has resulted in the development and maintenance of pedigreed Xiphophorus lines specifically bred for research. The recent availability of the X. maculatus reference genome assembly now provides unprecedented opportunities for novel and exciting comparative research studies among Xiphophorus species.
Results
We present sequencing, assembly and annotation of two new genomes representing Xiphophorus couchianus and Xiphophorus hellerii. The final X. couchianus and X. hellerii assemblies have total sizes of 708 Mb and 734 Mb and correspond to 98 % and 102 % of the X. maculatus Jp 163 A genome size, respectively. The rates of single nucleotide change range from 1 per 52 bp to 1 per 69 bp among the three genomes and the impact of putatively damaging variants are presented. In addition, a survey of transposable elements allowed us to deduce an ancestral TE landscape, uncovered potential active TEs and document a recent burst of TEs during evolution of this genus.
Conclusions
Two new Xiphophorus genomes and their corresponding transcriptomes were efficiently assembled, the former using a novel guided assembly approach. Three assembled genome sequences within this single vertebrate order of new world live-bearing fishes will accelerate our understanding of relationship between environmental adaptation and genome evolution. In addition, these genome resources provide capability to determine allele specific gene regulation among interspecies hybrids produced by crossing any of the three species that are known to produce progeny predisposed to tumor development.
Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Same of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern Iod from the platyfish Xiphophorus maculatus the melanoma-inducing gene has been doned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanomapredisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a geneduplication event, thereby acquiring the oncogenic potential. The homology of the melanomainducing Iod points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.
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 xmrkB or the non-tumorigenic xmrkA allele was performed. I was able to demonstrate that expression of the tumorigenic xmrkB 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 xmrkA allele, in contrast, is not influenced by the presence or absence of R. These findings strongly indicate that differential transcriptional regulation of the xmrk promoter triggers the tumorigenic potential of these xmrk alleles. To functionally characterize the xmrk promoter region, I established a luciferase assay using BAC clones containing the genomic regions where xmrk and egfrb are located for generation of reporter constructs. This approach showed for the first time a melanoma cell specific transcriptional activation of xmrkB by its flanking regions, thereby providing the first functional evidence that the xmrk oncogene is controlled by a pigment cell specific promoter region. Subsequent analysis of different deletion constructs of the xmrkB BAC reporter construct strongly indicated that the regulatory elements responsible for the tumor-inducing overexpression of xmrkB in melanoma cells are located within 67 kb upstream of the xmrk oncogene. Taken together, these data indicate that melanoma formation in Xiphophorus is regulated by a tight transcriptional control of the xmrk oncogene and that the R locus acts through this mechanism. As the identification of the R-encoded gene(s) is necessary to fully understand how melanoma formation in Xiphophorus is regulated, I furthermore searched for alternative R candidate genes in this study. To this end, three genes, which are located in the genomic region where R has been mapped, were evaluated for their potential to be a crucial constituent of the regulator locus R. Among these genes, I identified pdcd4a, the ortholog of the human tumor suppressor gene PDCD4, as promising new candidate, because this gene showed the expression pattern expected from the crucial tumor suppressor gene encoded at the R locus.
The observation of a slower migrating form of pp6oc-src in neural tissue of chicken and mouse has recently been shown to be due to an alternative transcript form of tbe c-src gene (Martinez et al.: Science 237:411-415, 1987; Levy et al.: Mol Cell Bio17:4142- 4145, 1987). An insertion of 18 basepairs between exons 3 and 4, presumed to be due to alternative splicing of a mini-exon, gives rise to six amino acid residues not found in the non-neuronal (termed flbroblastic) form of pp60\(^{c-src}\). Wehave addressed the question of the evolutionary origin of the c-src neuronal insert · and its functional signiflcance regarding neural-speciflc expression of the c-src gene. To this end we have investigated whether the c-src gene of a lower verlebrate (the teleost fish Xiphophorus) gives rise to a neural-specific transcript in an analogous manner. We could show that the fish c-src gene does encode for a "fibroblastic" and a "neuronal" form of transcript and that the neuronal transcript does indeed arise by way of alternative splicing of a mini-exon. The miniexon is also 18 basepairs long and we could demoostrate directly that this exon lies within the intron separating exons 3 and 4. For comparative purposes we have examined whether the fish c-yes gene, the member of the src gene family most closely related to c-src, also encodes a neural tissue-specific transcript. No evidence for a second transcript form in brain was obtained. This result suggests that the mini-exon arose within the c-src gene lineage sometime between the srclyes gene duplication event and the divergence of the evolutionary lineage giving rise to the teleost fish. Published genomic sequence of src-related genes in Drosophila and our own results with Hydra demoostrate no intron in these species at the analogous location, consistent with first appearance of this mini-exon sometime between 550 and 400 million years ago.
Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus
(2023)
Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.
Xiphophorus fish exhibit a clear phenotypic polymorphism in puberty onset and reproductive strategies of males. In X. nigrensis and X. multilineatus, puberty onset is genetically determined and linked to a melanocortin 4 receptor (Mc4r) polymorphism of wild-type and mutant alleles on the sex chromosomes. We hypothesized that Mc4r mutant alleles act on wild-type alleles by a dominant negative effect through receptor dimerization, leading to differential intracellular signaling and effector gene activation. Depending on signaling strength, the onset of puberty either occurs early or is delayed. Here, we show by Förster Resonance Energy Transfer (FRET) that wild-type Xiphophorus Mc4r monomers can form homodimers, but also heterodimers with mutant receptors resulting in compromised signaling which explains the reduced Mc4r signaling in large males. Thus, hetero- vs. homo- dimerization seems to be the key molecular mechanism for the polymorphism in puberty onset and body size in male fish.
Puberty is an important period of life with physiological changes to enable animals to reproduce. Xiphophorus fish exhibit polymorphism in body size, puberty timing, and reproductive tactics. These phenotypical polymorphisms are controlled by the Puberty (P) locus. In X. nigrensis and X. multilineatus, the P locus encodes the melanocortin 4 receptor (Mc4r) with high genetic polymorphisms.
Mc4r is a member of the melanocortin receptors, belonging to class A G-protein coupled receptors. The Mc4r signaling system consists of Mc4r, the agonist Pomc (precursor of various MSH and of ACTH), the antagonist Agrp and accessory protein Mrap2. In humans, MC4R has a role in energy homeostasis. MC4R and MRAP2 mutations are linked to human obesity but not to puberty.
Mc4rs in X. nigrensis and X. multilineatus are present in three allele classes, A, B1 and B2, of which the X-linked A alleles express functional receptors and the male-specific Y-linked B alleles encode defective receptors. Male body sizes are correlated with B allele type and B allele copy numbers. Late-maturing large males carry B alleles in high copy number while early-maturing small males carry B alleles in low copy number or only A alleles. Cell culture co-expression experiments indicated that B alleles may act as dominant negative receptor mutants on A alleles.
In this study, the main aim was to biochemically characterize the mechanism of puberty regulation by Mc4r in X. nigrensis and X. multilineatus, whether it is by Mc4r dimerization and/or Mrap2 interaction with Mc4r or other mechanisms. Furthermore, Mc4r in X. hellerii (another swordtail species) and medaka (a model organism phylogenetically close to Xiphophorus) were investigated to understand if the investigated mechanisms are conserved in other species.
In medaka, the Mc4r signaling system genes (mc4r, mrap2, pomc, agrp1) are expressed before hatching, with agrp1 being highly upregulated during hatching and first feeding. These genes are mainly expressed in adult brain, and the transcripts of mrap2 co-localize with mc4r indicating a function in modulating Mc4r signaling. Functional comparison between wild-type and mc4r knockout medaka showed that Mc4r knockout does not affect puberty timing but significantly delays hatching due to the retarded embryonic development of knockout medaka. Hence, the Mc4r system in medaka is involved in regulation of growth rather than puberty.
In Xiphophorus, expression co-localization of mc4r and mrap2 in X. nigrensis and X. hellerii fish adult brains was characterized by in situ hybridization. In both species, large males exhibit strikingly high expression of mc4r while mrap2 shows similar expression level in the large and small male and female. Differently, X. hellerii has only A-type alleles indicating that the puberty regulation mechanisms evolved independently in Xiphophorus genus. Functional analysis of Mrap2 and Mc4r A/B1/B2 alleles of X. multilineatus showed that increased Mrap2 amounts induce higher cAMP response but EC50 values do not change much upon Mrap2 co-expression with Mc4r (expressing only A allele or A and B1 alleles). A and B1 alleles were expressed higher in large male brains, while B2 alleles were only barely expressed. Mc4r A-B1 cells have lower cAMP production than Mc4r A cells. Together, this indicates a role of Mc4r alleles, but not Mrap2, in puberty onset regulation signaling. Interaction studies by FRET approach evidenced that Mc4r A and B alleles can form heterodimers and homodimers in vitro, but only for a certain fraction of the expressed receptors. Single-molecule colocalization study using super-resolution microscope dSTORM confirmed that only few Mc4r A and B1 receptors co-localized on the membrane. Altogether, the species-specific puberty onset regulation in X. nigrensis and X. multilineatus is linked to the presence of Mc4r B alleles and to some extent to its interaction with A allele gene products. This is reasoned to result in certain levels of cAMP signaling which reaches the dynamic or static threshold to permit late puberty in large males.
In summary, puberty onset regulation by dominant negative effect of Mc4r mutant alleles is a special mechanism that is found so far only in X. nigrensis and X. multilineatus. Other Xiphophorus species obviously evolved the same function of the pathway by diverse mechanisms. Mc4r in other fish (medaka) has a role in regulation of growth, reminiscent of its role in energy homeostasis in humans. The results of this study will contribute to better understand the biochemical and physiological functions of the Mc4r system in vertebrates including human.