Strukturelle und funktionelle Analyse ausgewählter High Mobility Group Gene des Haushundes

Medical advances due to scientific research made remarkable progress in the past decades fighting cancer. Nevertheless according to the United Nation s World Cancer Report 2008 tumour diseases are the leading cause of death with 7.6 million deaths and 12.4 million the new cancer cases a year. According to cancer the dog as an animal model joins the popular rodent model in order to study and understand the molecular genetics of tumour diseases and to develop new therapeutic strategies against cancer. Canine tumour diseases are distinguished by spontaneous development and dogs and humans share striking similarities in the case of oncology. The main part of this thesis comprises the comparative genomics and tumour biology of the human and canine tumour diseases focusing in particular prostate carcinomas by molecular characterisation of the genes and proteins HMGA1, HMGA2, THADA, RAGE, TNFalpha, and IL-alpha/beta. Genomic characterisations of high evolutionary conserved genes are the fundamentals for functional and structural analysis of tumour relevant genes. According to this principle a gene therapeutic approach was developed in order inhibit the HMGA expression of a canine prostate carcinoma cell line with the help of an adeno-associated virus vector. Furthermore and alternative method with gold nanoparticles was established for efficient transfection of eukaryotic cells. In the context of inflammation and angiogenesis during a tumour disease the canine receptor for advanced glycation end products (RAGE) was analysed. The results showed highly conserved molecular similarities (N.B. ligand-receptor domains) between the human and canine RAGE and its isoforms. In both species an aberrant Expression of cytokines e.g. interleukin-1 (IL-1alpha/beta) and the tumour necrosis factor alpha (TNFalpha) plays an important role during the initiation of immune response and inflammation. In particular during the course of the Langerhans cell histiocytosis (LCH) large quantities of cytokines are produced. Interestingly most clinical and pathological features of the canine malignant histiocytosis resemble those of LCH in humans. Finally dogs could be used as an animal model to elucidate the molecular genetics of malignant histiocytosis as well as LCH. In this connection nucleic base pair substitution within evolutionary conserved gene and amino acid sequences of canine and human cytokines were investigated

Regulation Of Fshr And SF-1 In The Hypothalamus-Pituitary-Gonadal (HPG) Axis.

Mammalian reproduction is highly dependent on the delicate balance of signals within the hypothalamic-pituitary-gonadal (HPG) axis that maintains proper endocrine environment. One of the key signals is the pituitary glycoprotein follicle-stimulating hormone (FSH) which is critical for gonadal development and fertility. FSH acts via FSHR, a G protein-coupled receptor present on a few distinct cell types, prominently on the Sertoli cells of the testis and granulosa cells of the ovary Prior studies have identified several key regulatory elements required for Fshr transcription but a clear understanding of what controls its limited cell specific expression remains elusive. Comparative genomics identified a number of evolutionary conserved regions (ECR) distal to the proximal promoter, indicating these regions might harbor regulatory elements. One such distal regulatory element known to play a role in transcriptional regulation is the "multivalent" protein CCCTC-binding factor (CTCF). Computational analysis of ECRs identified multiple CTCF binding sites in the intergenic regions of the Fshr locus and depletion of CTCF in rat granulosa cells led to a two-fold increase in Fshr mRNA. These data indicate that CTCF either by itself or in conjunction with other protein complexes might play a role in transcriptional regulation of the Fshr gene. Another important component of the HPG axis is the transcription factor, Steroidogenic factor-1 (SF-1) transcribed from the Nr5a1 gene (also known as Ftz-F1 or Ad4bp) which plays a pivotal role in adrenal and gonadal development and regulates genes at all levels of the axis. Comparative genomics identified two conserved sequences associated with Ftz-F1. One was found to encode a long non-coding RNA and the other a regulatory element important for SF-1 expression in the pituitary. A long non-coding RNA named Fast or Ftz-F1 associated transcript was found transcribed in the opposite direction to SF-1, co-expressed, and co-regulated with SF-1 and hormonally regulated. Knockdown and over-expression of the Fast transcript in MA-10, mouse Leydig cell line, did not alter the transcription or translation of SF-1. Long non-coding RNA have been focus of intense research and have been shown to play diverse role in various physiological and pathological processes. Considering their potential for transcriptional regulation, the identified long non-coding RNA, Fast, might play potentially important role in endocrine development and homeostasis. Comparative sequence analysis identified an evolutionary conserved region, ECR3, approximately 4kb away from the transcriptional start site of SF-1. Transient transfection data revealed ECR3, upregulated SF-1 transcription in alpha T3 (gonadotrope cell line) and Y-1 (adrenal) cells, but downregulated SF-1 in MA-10 (Leydig cell line), MSC-1 (Sertoli cell line) and primary rat peritubular Myoid and Sertoli cells. This region was found to contain an E-box and was bound by upstream stimulatory factors 1 and 2 (USF1 and USF2) and a basic-helix-loop-helix (bHLH) protein E2A. In particular, co-transfection studies identified Inhibitors of DNA binding, Id2 and Id3 to downregulate SF-1 transcription. In summary, this dissertation looks at the regulation of Fshr and SF-1 in the HPG axis and identifies the importance of distal regulatory sequences and non-coding transcripts and its role in gene regulation

Non-Canonical cis-Acting Elements Regulating Polyadenylation of the Beta-Adducin Pre-mRNA

The beta adducin gene has a very complex architecture, with tissue-specific use of promoters and polyadenylation sites (PAS). Its expression is restricted to neuronal and hematopoietic tissues. In mice, usage of a distal brain-specific PAS (PAS4) generates an 8. 3 kb long mRNA with an unusually long 3’UTR of about 5.7 kb. Instead, the use of proximal erythroid-specific PASs results in short 3.1-3.7 kb mRNA isoforms. The presence and use of the different alternative PASs in the beta adducin gene are very well conserved among species suggesting an important and specific role for each of the generated 3’UTRs. To study the regulatory mechanisms involved in beta adducin polyadenylation, minigene constructs containing the mouse beta adducin polyadenylation signals were used. The constructs were transfected into HeLa cells and their expression and PAS selection determined by Northern blot analysis. The exclusive usage of the PAS4 in HeLa cells has been observed. All the elements defining the core polyadenylation signal were characterized: the hexanucleotide motif (Hm), the upstream and downstream sequence elements (USE and DSE, respectively) elements, and the cleavage site. Interestingly, I have detected the presence of two novel non-canonical cis-acting elements regulating 3’end processing at the PAS4. Both elements act on long-distance and to the best of our knowledge, long-distance upstream polyadenylation regulatory elements have not previously been described for the non-viral eukaryotic transcripts. The first of these elements was essential to enable polyadenylation at the PAS4. It was located in a region spanning 355 nucleotides and includes the stop codon of beta adducin. The second non-canonical upstream polyadenylation regulatory element seems to inhibit processing at the PAS4. It was located in the region close to the second proximal PAS, about 4.5 kb upstream of the PAS4. These results highlight the complexity of the regulatory mechanisms directing beta adducin pre-mRNA 3’end processing

Regulation of Emx2 expression by antisense transcripts in the murine developing CNS

In this thesis I studied the expression pattern of Emx2OS, the antisense partner of the homeobox gene Emx2, in the developing mouse nervous system. Emx2OS was detectable in telencephalon, mammillary recess, mesencephalon, nasal pits and otic vesicle, all of them also expressing Emx2. Within dorsal telencephalon, Emx2OS peaked in post-mitotic neurons, specifically at the time when they completed radial migration and turned Emx2 off. Such pattern suggested that Emx2OS may be implicated in regulation of Emx2, according to complex and even antithetic ways. By artificially modulating Emx2OS in primary cortico-cerebral precursors, via lentiviral RNAi and somatic transgenesis, we found that such transcript contributes to down-regulation of its sense partner, possibly by a Dicer-dependent posttranscriptional mechanism. On the other side, by ectopically activating Emx2OS in primary rhombo-spinal precursors, we elicited a robust activation of Emx2. Further inspection of Emx2 null cortices conversely showed a collapse of Emx2OS expression. Taken together, these results suggest that a mutual positive loop involving Emx2 and Emx2OS is necessary to adequate expression of either transcript in the early neural tube

XDazl function in RNA metabolism in Xenopus laevis

Germ cell specification in Xenopus laevis depends on the inheritance of maternal determinants from the oocyte in form of mRNAs and proteins. At the onset of zygotic transcription, maternal mRNAs are subjected to miR-mediated decay. Interestingly, germ cell specific mRNAs are excluded from miR-induced degradation. Recent studies have provided insights into how RNA-binding proteins can modulate miR-mediated regulation of RNA stability. In Xenopus embryos for example, ElrB1 and Dead end (XDE) proteins have been demonstrated to protect germ cell specific mRNAs from miR-mediated clearance. Recognition of these mRNAs by miRs relies on target sites within the localization element (LE), a specific region in the 3’ UTR, which is also required for vegetal mRNA transport in the oocyte (Koebernick et al. 2010). In this study, we addressed the role of Deleted in azoospermia-like (XDazl) in the context of germline specific transcript stabilization in Xenopus embryos. The RNA binding protein XDazl is provided maternally and its zygotic transcription starts at stage 17. Ectopic expression of XDazl promotes stabilization of the PGC-specific XDE mRNA as well as that of other germline transcripts. In vitro RNA binding assays indicate direct interaction of XDazl with these transcripts. Moreover, a synergistic effect of XDE and XDazl protein in stabilizing germ cell specific mRNAs was observed. Mapping of the XDazl binding site within the XDE-LE indicated overlapping binding sites with XDE and ElrB1. Degradation of germ cell specific mRNAs is mediated by miRs, since blocking of miR processing leads to enrichment of germline transcripts. XDazl protein binds directly to the LE in its own mRNA, which contains at least three independent XDazl binding sites. The knockdown of XDazl in embryos leads to a reduced number of germ cells at tailbud stage, indicating an important role of XDazl during germ cell formation or maintenance. Immunofluorescence staining of oocytes shows XDazl protein enrichment at the vegetal cortex and in transport particles colocalizing with XDE and XDazl mRNAs, suggesting an early involvement of XDazl in the context of vegetal mRNA transport and/or anchoring