Medicine (RMH) - Theses
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ItemCharacterisation of the nuclear pore abnormalities in the intestinal zebrafish mutant, flotte lotte (flo)( 2012)The evolution of eukaryotic cells is defined by the compartmentalisation of the genetic material inside the nucleus, segregated from cytoplasm by a nuclear envelope. This barrier is punctuated by approximately 3000 large multi-protein structures known as nuclear pore complexes, which permit the bidirectional transport of protein and RNA molecules between the nucleus and cytoplasm. This study provided the opportunity to investigate the importance of the nuclear pore protein Elys during vertebrate development. Zebrafish mutants generated by ethylnitrosourea (ENU) mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes. Our interest has focused on a panel of ENU generated mutants exhibiting a variety of defects in the formation and differentiation of the intestinal epithelium. One of these mutants, flotte lotte (flo), harbours a premature stop codon in the coding sequence of the nuclear pore component elys (embryonic large molecule derived from yolk sac). Elys is an essential component of the nuclear pore complex, yet surprisingly, its mutation in the flo mutant does not result in a global dysfunction in nuclear pore formation throughout the developing zebrafish embryo. Instead, flo mutants exhibit tissue-specific abnormalities in the development of the intestinal epithelium, liver, pancreas and eye; organs that are highly proliferative from 48hpf. We show that this time-point coincides with the exhaustion of maternally-deposited stocks of elys mRNA from flo embryos. Not surprisingly, we found that the ensuing inability to create new nuclear pore complexes appears to impact most severely on these rapidly proliferating tissues. Using multi-photon microscopy we reconstructed three-dimensional renditions of the endodermal organs in wild-type and flo larvae. Compared to the highly elaborated and polarized intestinal epithelium of wild-type zebrafish, the intestinal epithelium in flo is thin, unfolded and poorly polarised. Moreover, nuclear pore complexes in flo intestinal cells are not embedded in the nuclear envelope but are found in profuse cytoplasmic aggregates. Catastrophic levels of apoptosis accompany the loss of a functional nuclear envelope in intestinal epithelial cells. Thus, flo mutants provide an opportunity to identify signals that commit nuclear pore-deficient cells to an apoptotic fate. We found that the apoptotic response observed in the flo intestine is mediated via a Tp53-independent mechanism. Since Elys function is critical for the integrity of proliferative cells in zebrafish, we investigated whether ELYS is also critical for the proliferation of human cancer cells. We discovered a strong up-regulation of ELYS expression in many cancers when compared to their respective normal tissues. We observed ELYS to be ranked in the top 1% of all up-regulated genes investigated in gene expression studies of colorectal, kidney, liver and breast cancers available in the Oncomine database. The discovery that ELYS is frequently over-expressed in human colorectal cancer suggests that our functional genomics approach to novel cancer gene discovery using zebrafish mutants is valid. Moreover, we propose that targeted approaches to disabling ELYS synthesis or function may activate apoptosis in colorectal cancer cells and provide a useful therapeutic approach in the future.
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ItemThe role of the transcription/repair factor TFIIH in the intestinal dysplasia of the zebrafish mutant sycorax( 2012)The gene expression profiles of various cancer types show significant similarities to those of their developing tissue of origin, with a number of genes that are expressed in the developing intestine but not the adult intestine being re-expressed in colorectal cancer (CRC) cells. This study utilised a zebrafish intestinal mutant, sycorax, to further the current understanding of the genetics underlying vertebrate intestinal development and development and progression of CRC. The sycorax zebrafish mutant displays a dysplastic intestinal epithelium, with multilayering and disorganisation, reminiscent of a pre-neoplastic lesion. The mutation underlying the sycorax phenotype is a premature stop codon in the general transcription 2 H, polypeptide 4 (gtf2h4) gene, which is predicted to generate a truncated Gtf2h4 protein. Gtf2h4 is one of the components of the multisubunit complex, general transcription factor IIH (TFIIH), which is integral to a number of cellular processes. TFIIH is one of the general transcripton factors involved in transcription initation of all protein coding genes via RNA polymerase II. It also fine tunes the expression of a small proportion of genes by integrating signals from gene specific activators and repressors. TFIIH is involved in the regulation of transcription of rRNA by RNA polymerase I, in addition to being a crucial component of the DNA repair pathway known as nucleotide excision repair. Finally, TFIIH plays a role in the regulation of cell cycle progression. Northern blot analysis and metabolic labeling of newly synthesized rRNA in wild type and sycorax embryos demonstrated an increase in rRNA transcription in sycorax embryos. sycorax embryos were also shown to have reduced DNA repair capabilities following UV irradiation. Increased levels of proliferation in the intestine, as demonstrated by EdU incorporation, are hypothesised to contribute to the multilayering of the sycorax intestinal epithelium. Gene expression analysis of wild type and sycorax intestines identified a number of differentially expressed genes, many of which are involved in the regulation of cell proliferation. Upregulated genes in the sycorax intestine include c-myc and c-myb, which have previously been shown to be directly transcriptionally regulated by TFIIH, and are proposed to contribute to the increased levels of proliferation observed in the sycorax intestine. Furthermore, knockdown of c-myb in sycorax embryos rescues the intestinal dysplasia. These results demonstrate that sycorax embryos display abnormalities in all the processes in which TFIIH plays a role; therefore, the sycorax mutant provides a novel opportunity to further study the function of TFIIH in an entire vertebrate organism. Strikingly, as the sycorax intestinal phenotype is reminiscent of a pre-neoplastic lesion, and the oncogenes c-myc and c-myb are upregulated in the sycorax intestine, this study has identified a possible role for altered TFIIH function in CRC.
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ItemThe U12-dependent spliceosome is essential for regulating gene expression during zebrafish development( 2010)Removal of introns from pre-mRNA is an essential step in generating mature mRNA. The majority of introns are removed by the major, or U2-type, spliceosome, while the minor, or U12-type, spliceosome catalyses the removal of a small set of introns with characteristic features that are highly conserved in metazoans and plants. A novel zebrafish mutant, caliban (cal), with specific U12-type splicing defects, was used to study the biochemistry of the U12-type spliceosome and the role of U12-type introns in the regulation of gene expression. Greater than 99% of vertebrate introns are U2-type introns. However, a second class of U12-type introns exists that is defined by highly conserved consensus splice sites. U12-type introns represent about 0.5% of all introns in vertebrate genomes and usually occur alone alongside U2-type introns in pre-mRNA. U12-type introns are removed by a separate spliceosome that shares many components with the U2-type spliceosome, but contains several unique small nuclear RNAs (snRNAs) and spliceosomal proteins. Several lines of evidence suggest a function for U12-type splicing in the regulation of gene expression. These include the high evolutionary conservation of U12-type introns, their enrichment in certain functional gene groups and the demonstration that their excision can be rate-limiting in the generation of mature mRNAs. Despite these observations, little is known about the role and possible regulatory function of U12-type splicing in vivo. cal is a zebrafish development mutant with abnormalities in the intestinal epithelium, which is poorly polarised and unfolded compared to wildtype (wt). Mutant embryos also show a reduction in size of the liver and the pancreas and display a morphologically abnormal lens in the context of a smaller eye. The genetic lesion in cal was mapped to rnpc3, encoding the zebrafish orthologue of the human U11/U12 snRNP 65KDa protein, a specific component of the U12-type spliceosome. It was shown that cal embryos specifically retain U12-type introns compared to wildtype. Biochemical analyses of U12-type spliceosomal small nuclear ribonucleoproteins (snRNPs) demonstrate abnormal formation of the U11/U12 di-snRNP, which represents the first step in U12-type spliceosome assembly. However, it was also demonstrated that larger spliceosomal particles form and accumulate in cal embryos in the absence of Rnpc3/65K, suggesting a potential novel role for Rnpc3/65K in U12-type spliceosome disassembly and recycling. Whole transcriptome analysis of cal and wt embryos by microarrays and RNA sequencing demonstrated retention of U12-type introns on a global scale as well as a set of about 700 differentially expressed genes between cal and wt at two different developmental time points. Further analysis of gene expression data has led to the emergence of a model in which cal embryos are sustained through the first 48-72hpf by maternally deposited rnpc3 mRNA and Rnpc3/65K protein. After this time, defective U12-type splicing induces a cell cycle arrest in the endoderm-derived tissues of the liver, pancreas and intestine, which are highly proliferative between 72 and 108hpf. These results are leading to further studies with a focus on the role of U12-type splicing in human cancer. It was found that several prominent human tumour suppressor genes such as PTEN, LKB1 and PROX1, contain U12-type introns, and we propose a model by which an intermediate reduction of U12-type splicing efficiency can be tumourigenic by reducing the activity of particular tumour suppressor genes in a dose-dependent fashion. To test this hypothesis, conditional Rnpc3 knockout mouse models are currently being generated on a range of different colorectal cancer-susceptible backgrounds.