Environment Institute Leaders publications

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Browsing Environment Institute Leaders publications by Advisors "Grutzner, Frank"

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    A first look at monotreme meiotic recombination.
    (2012) Horan, Sonia; Grutzner, Frank; Daish, Tasman James; Tsend-Ayush, Enkhjargal; School of Molecular and Biomedical Science
    Recombination is a critical event in meiosis required for proper chromosome segregation; however, despite its importance, crossovers have not yet been analysed in egg-laying mammals, representing an extant species from the most basal of the contemporary mammalian lineage. The platypus is a unique species in that it has a complex system of ten sex chromosomes that form a chain in meiosis, and the segregation of this chain raises many questions regarding the state of meiotic recombination among chain members. This study aimed to set a foundation for recombination studies in the platypus. 15 genes essential for crossover formation were analysed; while most of these genes showed generally high levels of conservation, surprisingly it was found that Mei4 and Rec114 are not expressed during platypus meiosis. This raises several questions about the platypus double-strand break induction mechanism in which these genes have an essential function in other mammals. Furthermore, characterisation of the crossover hotspot-determining protein, Prdm9, shows that the platypus protein differs significantly from the Prdm9 of eutherians, suggesting that the gene underwent rapid evolution after the divergence of monotremes nearly 200 Million years ago. This study also aimed to examine the distribution of crossovers in meiotic cells using Mlh1 as a marker for crossover events. Despite conservation of the protein and expression and some specific staining overall, crossover counts could not be analysed in platypus male meiotic cells. Surprisingly another protein which can be found in earlier meiotic stages, Dmc1, showed a very distinct pattern in platypus pachytene cells. It was observed that an average of 24 Dmc1 foci were present in platypus pachytene, long after Dmc1 is expected to have diminished from chromosome cores. These foci seemed to localise to the ends of chromosomes and were more prominently associated with the sex chromosomes, particularly the pseudoautosomal regions. Together with other evidence this suggests that the Dmc1 antibody may be cross-reacting with Rad51, which plays a structural role in pachytene, holding chromosomes together at the telomeres. Overall this work provides first evidence for a general conservation of genes involved in the recombination pathway but has also identified some genes that may have diversified in their function.
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    Function and evolution of the piRNA pathway in the amniote gonad and human ovarian cancer.
    (2012) Lim, Shu Ly; Grutzner, Frank; Tsend-Ayush, Enkhjargal; School of Molecular and Biomedical Science
    The Piwi-interacting RNA (piRNA) pathway is a RNA silencing pathway which represses the expression of gene and transposable elements (TE) in the gonads via the binding of piRNAs (26-30nt) to their complimentary RNA targets and by influencing the epigenetic makeup of chromatin via interacting with other proteins (e.g. HP1). piRNAs interact with PIWI (P-element induced wimpy testis) proteins and other components such as Maelstrom (Mael) for TE silencing. In addition, Piwi-like (Piwil) genes and Mael are important for germline stem cell (GSC) production from fly to mouse. The expression of these genes was reported exclusively in mammalian testis despite the presence of piRNAs in mouse testis and ovary. Although the pathway is essential for oogenesis in fly, fish and Xenopus, an important role in the mammalian ovary has been in doubt, as female knockout mice (Piwil1, 2 and Mael) genes are fertile. In addition Piwil genes in particular have undergone lineage specific changes leading to up to 4 Piwil genes in most eutherian mammals. Work presented in this thesis investigates the expression of piRNA pathway genes in the amniote gonad. This confirmed the robust expression of Piwil genes and Mael in the mammalian testis. Importantly, specific expression of these genes in oocyte and growing follicles was detected in mammals. The extraordinary conservation of piRNA pathway gene expression in germ cells and ovarian somatic cells from fly to human suggests an important role in mammalian gonadal development. A comprehensive bioinformatics analysis of Piwil genes provided new aspects towards understanding the evolutionary trajectory of Piwi like genes in vertebrates. For example the correction of Xenopus piwil3 as piwil1 ortholog clearly showing that Piwil3 evolved exclusively in eutherian mammals. Finally, based on the expression of piRNA pathway genes in the ovary, we hypothesised that PIWIL genes and MAEL may play a part in the origin and progression of epithelial ovarian cancer (EOC). To test this hypothesis, expression of these genes was investigated in postovulatory tissues i.e. the corpus luteum (CL) and inclusion cysts. Preliminary data show that piRNA pathway genes are not expressed in the CL but transcripts are detected in the epithelial cells of inclusion cysts. This raises the possibility that piRNA pathway genes may be involved in the cancerous transformation of epithelial cells. To test if the piRNA pathway plays a part in EOC progression, and is related to the activity of TEs, the expression of PIWIL genes, MAEL and L1 (one of the most abundant TEs in human) was investigated in different types of EOC. Significant upregulation of these genes was found in malignant EOC when compared to benign ovarian tumours. This upregulation might be a result of increased L1 activity in EOC, or may due to the stem cell like characteristic of malignant EOC. Analyses of the PIWIL1 transcripts from a malignant EOC show that most of the PIWIL1 transcripts contain premature stop codons. Therefore, although PIWIL1 is overexpressed in malignant EOC, the function of PIWIL1 is likely to be compromised in these tumours. To understand the effects of PIWIL1 and MAEL overexpression on cancer cell invasiveness, PIWIL1 and MAEL were transiently overexpressed in ovarian cancer SKOV3 cells. Overexpression of these genes decreases cell invasiveness, suggesting a repressive role in EOC progression. Hypomethylation of L1 and chromosome instability was found in ovarian cancers. To understand the genome stability in EOC, primary ovarian cancer cells were established from patient ascites of different stages of ovarian cancers. FISH analyses showed that 20%-40% of the cells are aneuploid. Thus, this is a good model for understanding aneuploidy in EOC development. Our research provides a better understanding of this ancient yet conserved piRNA pathway in mammalian gonads and ovarian cancers.