@phdthesis{Regneri2013,
  author    = {Regneri, Janine},
  title     = {Transcriptional regulation of cancer genes in the Xiphophorus melanoma system},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-82319},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  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 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.},
  subject      = {Melanom},
  language  = {en}
}
@phdthesis{Carstensen2018,
  author    = {Carstensen, Anne Carola},
  title     = {Identification of novel N-MYC interacting proteins reveals N-MYC interaction with TFIIIC},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143658},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated. To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif. Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin.},
  subject      = {Biologie},
  language  = {en}
}