The xmrk oncogene can escape nonfunctionalization in a highly unstable subtelomeric region of the genome of the fish xiphophorus☆

doi:10.1016/S0888-7543(03)00168-X

Genomics

Volume 82, Issue 4, October 2003, Pages 470-479

https://doi.org/10.1016/S0888-7543(03)00168-X Get rights and content

Abstract

The Xmrk oncogene involved in melanoma formation in the fish Xiphophorus was formed relatively recently by duplication of the epidermal growth factor co-orthologue egfrb. In the platyfish X. maculatus, Xmrk is located close to the major sex-determining locus in a subtelomeric region of the X and Y sex chromosomes that frequently undergoes duplications and other rearrangements. This region accumulates repetitive sequences: more than 80% of the 33-kb region 3′ of Xmrk is constituted by retrotransposable elements. The high degree of nucleotide identity between X- and Y-linked sequences and the rarity of gonosome-specific rearrangements indicated that the instability observed was not a manifestation of gonosome-specific degeneration. Seven other duplicated genes were found, all corresponding, in contrast to Xmrk, to pseudogenes (nonfunctionalization). Functional persistence of Xmrk in a highly unstable region in divergent Xiphophorus species suggests a beneficial function under certain conditions for this dispensable and potentially injurious gene.

Section snippets

Preferential accumulation of transposable elements in the Xmrk oncogene region of X. maculatus

Analysis of the genomic sequence flanking the Y allele of the platyfish Xmrk gene on its 5′ side (49 kb; Fig. 1) and its 3′ side (71 kb; Fig. 2) revealed the presence of diverse categories of transposable elements and other repetitive sequences (Table 1). Strikingly, more than 80% (82.2%) of the 33-kb genomic region flanking Xmrk 3′ of its polyadenylation signal is constituted by retrotransposable elements, particularly LTR retrotransposons (Fig. 2). Xmrk is properly encircled by repetitive

Discussion

Gene duplication plays an important role in the evolution of gene regulation and gene function. The Xmrk region frequently undergoes duplications as well as other kinds of rearrangements, which can potentially lead to exon shuffling or to the recruitment of new regulatory sequences. Hence, despite a high frequency of miscarriage through nonfunctionalization, the genomic plasticity observed might allow the appearance of evolutionary innovations in the Xmrk region, which is therefore an excellent

Fishes and DNA libraries

X. maculatus strain WLC1274 (population Rio Jamapa; Veracruz, Mexico) and X. montezumae strain WLC1052 (Cascadas de Tamasopo, San Luis Potosi, Mexico) were kept at the Biozentrum of the University of Würzburg (Germany). Xmrk- and egfrb-containing clones were isolated from XY cosmid and BAC genomic libraries [44], [45].

DNA analysis

Southern blot hybridization was performed in 50% formamide at 42°C, filters were washed with 1× SSC/1% SDS at 60°C. Xmrk- and egfr-containing genomic library clones were isolated

Acknowledgements

This work is supported by the Biofuture program of the German Bundesministerium für Bildung und Forschung (to J.N.V.), the Sonderforschungsbereich 465 of the Deutsche Forschungsgemeinschaft (to M.S. and J.N.V.), and the Fonds der Chemischen Industrie (to M.S.).

References (46)

A. Meyer et al.
Gene and genome duplications in vertebratesthe one-to-four (-to-eight in fish) rule and the evolution of novel gene functions
Curr. Opin. Cell. Biol.
(1999)
M. Schartl
Platyfish and swordtailsa genetic system for the analysis of molecular mechanisms in tumor formation
Trends Genet.
(1995)
A. Gomez et al.
Ligand-independent dimerization and activation of the oncogenic Xmrk receptor by two mutations in the extracellular domain
J. Biol. Chem.
(2001)
R. Poulter et al.
A LINE element from the pufferfish (Fugu) Fugu rubripes which shows similarity to the CR1 family of non-LTR retrotransposons
Gene
(1999)
R. Poulter et al.
A retrotransposon family from the pufferfish (Fugu) Fugu rubripes
Gene
(1998)
F.E. Pryde et al.
Chromosome endsall the same under their caps
Curr. Opin. Genet. Dev.
(1997)
J.-N. Volff et al.
A new beginning with new endslinearisation of circular chromosomes during bacterial evolution
FEMS Microbiol. Lett.
(2000)
M. Baudler et al.
Activation of transcription of the melanoma inducing Xmrk oncogene by a GC box element
J. Biol. Chem.
(1997)
C. Burgtorf
Gridded genomic libraries of different chordate speciesa reference library system for basic and comparative genetic studies of chordate genomes
Genomics
(1998)
A. Froschauer
Construction and initial analysis of bacterial artificial chromosome (BAC) contigs from the sex-determining region of the platyfish Xiphophorus maculatus
Gene
(2002)

J. Fischer et al.

The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66

Gene

(1996)

A. Amores

Zebrafish hox clusters and vertebrate genome evolution

Science

(1998)

J. Wittbrodt et al.

More genes in fish?

BioEssays

(1998)

M. Robinson-Rechavi

Euteleost fish genomes are characterized by expansion of gene families

Genome Res.

(2001)

J.-N. Volff et al.

Evolution of signal transduction by gene and genome duplication in fish

J. Struct. Funct. Genom.

(2003)

J.S. Taylor et al.

Comparative genomics provides evidence for an ancient genome duplication event in fish. Philos

Trans. R. Soc. London B Biol. Sci.

(2001)

M. Robinson-Rechavi et al.

An ancestral whole-genome duplication may not have been responsible for the abundance of duplicated fish genes

Curr. Biol.

(2001)

J. Wittbrodt

Novel putative receptor tyrosine kinase encoded by the melanoma-inducing Tu locus in Xiphophorus

Nature

(1989)

C. Wellbrock et al.

Signal transduction by the oncogenic receptor tyrosine kinase Xmrk in melanoma formation of Xiphophorus

Pigment Cell Res.

(1997)

S. Weis et al.

The macromelanophore locus and the melanoma oncogene Xmrk are separate genetic entities in the genome of Xiphophorus

Genetics

(1998)

F. Anders

Contributions of the Gordon–Kosswig melanoma system to the present concept of neoplasia

Pigment Cell Res.

(1991)

R.B. Walter et al.

Xiphophorus interspecies hybrids as genetic models of induced neoplasia

ILAR J.

(2001)

A. Schartl et al.

Spontaneous melanoma formation in nonhybrid Xiphophorus

Cancer Res.

(1995)

Cited by (25)

Transcriptional control analyses of the Xiphophorus melanoma oncogene
2015, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
Citation Excerpt :
The whole genomic region that harbors xmrk is a hotspot for transposon integrations, and thus, xmrk is encircled by diverse categories of transposable elements, other repetitive sequences and pseudogenes (Froschauer et al., 2001; Volff et al., 2003). While most of these elements are present at the same position in the xmrkA and xmrkB flanking regions, some elements, like for example a piggyBac-like insertion in the proximal xmrk 5′ flanking region, were shown to be allele specific (Volff et al., 2003). Hence, as (retro)transposons are targeted and often silenced by the epigenetic machinery of the host cell (Levin and Moran, 2011; Rigal and Mathieu, 2011), transposable element induced epigenetic variations leading to transcriptional differences between the two alleles are conceivable.
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 200 kb 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.
Expression regulation triggers oncogenicity of xmrk alleles in the Xiphophorus melanoma system
2012, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
Citation Excerpt :
The altered transcriptional regulation of the xmrk oncogene compared to egfrb is proposed to be due to a new 5′ upstream flanking region. It was shown that xmrk has acquired this new promoter region after the duplication event (Adam et al., 1993; Volff et al., 2003). Based on these data, we hypothesized that the tumor suppressing effect of the R locus on xmrk is exerted by directly or indirectly downregulating xmrk expression on the transcriptional level, and thus, R acts upstream of xmrk.
The Xiphophorus melanoma model has gained attention in biomedical research as a genetic model for tumor formation. Melanoma development 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 a so far unknown regulator locus R. To test the hypothesis that R is involved in transcriptional regulation of xmrk and consequently acts upstream of the xmrk signal, we performed a quantitative analysis of xmrk transcript levels in normal and melanoma tissues of different Xiphophorus genotypes carrying either a highly tumorigenic or a non-tumorigenic xmrk allele. Our results demonstrate that expression of the tumorigenic xmrk allele is highly increased in malignant melanomas compared to benign lesions, macromelanophore spots, and healthy skin. Transcription of the non-tumorigenic xmrk allele in pigment cells, in contrast, is not influenced by the presence or absence of R. These findings strongly indicate that differential transcriptional regulation of the xmrk promoter determines the tumorigenic potential of xmrk alleles in the Xiphophorus melanoma system, thereby supporting the hypothesis that R suppresses the oncogenic function of xmrk on the level of transcriptional control.
From Mendelian to molecular genetics: the Xiphophorus melanoma model
2006, Trends in Genetics
In human tumor biopsies it is almost impossible to pinpoint the particular molecular abnormalities that determine neoplasia. In animal models where tumorigenesis is initiated by clearly defined genetic events, it is possible to study the genes and their functions that make a normal cell become a fully malignant cancer cell. In the fish Xiphophorus, melanoma can be initiated by simple crossings, and the signaling pathways that govern tumor growth and progression can be delineated. This model offers the prospect of obtaining a complete picture of the molecular changes and regulatory networks underlying tumor formation, which should contribute to a better understanding of some general principles of cancer biology, and identify new targets for melanoma research in particular.
Genetic, biochemical and evolutionary facets of Xmrk-induced melanoma formation in the fish Xiphophorus
2004, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
Certain interspecific hybrids of the fish Xiphophorus spontaneously develop melanoma induced by the derepression of the Xmrk oncogene. Xmrk is a recent duplicate of an orthologue of the mammalian epidermal growth factor receptor gene Egfr. In addition to a specific overexpression in melanoma, amino-acid substitutions in the extracellular domain leading to ligand-independent dimerisation and constitutive autophosphorylation are responsible for the tumorigenic potential of Xmrk. The Xmrk receptor induces several signal transduction pathways mediating cell proliferation and resistance to apoptosis and initiating dedifferentiation. Moreover, Xmrk upregulates the expression of the secreted protein osteopontin, inducing an autocrine loop possibly allowing invasion and survival in the dermis as a first step in malignancy. Hence, Xmrk is able to induce pathways essential for a transformed phenotype. Some of these events are equivalent to those found downstream of the mammalian Egfr, but others have clearly evolved differently or are specific for pigment cells. Xmrk is potentially hazardous, nonessential and located in a very unstable genomic region. Nevertheless, Xmrk has been maintained under purifying selection in divergent Xiphophorus species. Hence, Xmrk has probably a beneficial function under certain conditions. The analysis of this function is a major challenge for future research in the Xiphophorus model.
The colorful sex chromosomes of teleost fish
2018, Genes
Dynamics of vertebrate sex chromosome evolution: from equal size to giants and dwarfs
2016, Chromosoma

View all citing articles on Scopus

^☆: Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under Accession Nos. AY228504, AY298856, AY298857, AY298858, and AY298859.

¹: Present address: Molecular Hematology, University of Frankfurt Medical School, D-60590 Frankfurt, Germany.

View full text

Regular articleThe xmrk oncogene can escape nonfunctionalization in a highly unstable subtelomeric region of the genome of the fish xiphophorus☆☆

Abstract

Section snippets

Preferential accumulation of transposable elements in the Xmrk oncogene region of X. maculatus

Discussion

Fishes and DNA libraries

DNA analysis

Acknowledgements

Curr. Opin. Cell. Biol.

Trends Genet.

J. Biol. Chem.

Gene

Gene

Curr. Opin. Genet. Dev.

FEMS Microbiol. Lett.

J. Biol. Chem.

Genomics

Gene

Gene

Zebrafish hox clusters and vertebrate genome evolution

Science

More genes in fish?

BioEssays

Euteleost fish genomes are characterized by expansion of gene families

Genome Res.

Evolution of signal transduction by gene and genome duplication in fish

J. Struct. Funct. Genom.

Comparative genomics provides evidence for an ancient genome duplication event in fish. Philos

Trans. R. Soc. London B Biol. Sci.

An ancestral whole-genome duplication may not have been responsible for the abundance of duplicated fish genes

Curr. Biol.

Novel putative receptor tyrosine kinase encoded by the melanoma-inducing Tu locus in Xiphophorus

Nature

Signal transduction by the oncogenic receptor tyrosine kinase Xmrk in melanoma formation of Xiphophorus

Pigment Cell Res.

The macromelanophore locus and the melanoma oncogene Xmrk are separate genetic entities in the genome of Xiphophorus

Genetics

Contributions of the Gordon–Kosswig melanoma system to the present concept of neoplasia

Pigment Cell Res.

Xiphophorus interspecies hybrids as genetic models of induced neoplasia

ILAR J.

Spontaneous melanoma formation in nonhybrid Xiphophorus

Cancer Res.

Regular article
The xmrk oncogene can escape nonfunctionalization in a highly unstable subtelomeric region of the genome of the fish xiphophorus☆☆