Nuclear transfer (NT), or cloning, which is the transfer of a donor nucleus to a recipient enucleated oocyte, has been successfully achieved to produce viable offspring in many species. The process is very inefficient, as reprogramming of the donor nucleus is required, and losses are high throughout development. Placentation abnormalities are a common feature amongst cloned animals. Incomplete nuclear reprogramming and erroneous epigenetic imprinting may contribute to aberrant protein transcription and DNA mutations, affecting mitochondrial metabolism and inducing cellular stress. In vitro produced embryos under high oxygen culture conditions may also suffer oxidative stress, with the resulting reactive oxygen species causing mitochondrial DNA mutations and cellular stress similar to clones. In this study, expression of oxidative stress protein markers (Hsp60, SOD2, Hsp70) in NT cotyledons were compared to artificial insemination (AI) at different time points of gestation (days 50, 100, and 150). As a continuum of the oxidative stress investigation in cloned cotyledons, in vitro produced embryos were cultured under 20% oxygen compared to the control 7% oxygen laboratory standard culture, with oxidative stress protein markers examined between the groups at blastocyst stage (day 7) and day 15. Embryo morphology was also observed to determine apparent physiological differences between the treatment and control embryos. No previous studies to date have investigated the developmental effects of oxidative stress in day 15 bovine embryos. The significant differences in oxidative stress proteins observed at several time points in the NT and AI groups were not repeatable, possibly due to sample freeze/thaw degradation. Morphological differences observed between embryos cultured in 20% oxygen and control groups were visually apparent, although not quantified. At day 15 manganese superoxide dismutase expression was significantly lower in the 20% group compared to control. The 20% oxygen group did not show higher heat shock protein 60 expression than control, however the same results have been observed in another study at blastocyst stage. The results of this study suggest that the effect of oxidative stress on embryonic development is evident yet inconclusive in bovine NT cotyledons, however does not appear apparent in day 15 embryos following culture in 20% oxygen.

Après intégration dans le génome cellulaire de l’hôte, l’expression des rétrovirus dépend d’éléments agissant en cis localisés dans la longue répétition terminale 5’ (LTR5’) et la région leader, de facteurs de transcription cellulaires et viraux agissant en trans ainsi que de l’organisation chromatinienne du provirus intégré. Notre laboratoire a précédemment identifié dans le génome du rétrovirus HIV-1 (Human Immunodeficiency Virus type 1) une région intragénique importante (nt 4079-6026, où nt +1 est le début de U3 dans le LTR5’) composée du fragment 5103, du site hypersensible aux nucléases SH7 et du fragment 5105. Lors de ce travail, nous avons caractérisé physiquement et fonctionnellement différents sites de liaison pour des facteurs de transcription cellulaires localisés dans la région intragénique du virus HIV-1, dont trois sites de liaison pour le facteur inductible AP-1, dans des expériences de retard de migration sur gel et de transfection transitoire. Nous avons montré l’importance de ces trois sites AP-1 pour la réplication virale au niveau transcriptionnel dans des expériences d’infection et d’immunoprécipitation de la chromatine. De plus, nous avons caractérisé l’activité transcriptionnelle associée à la région intragénique du virus HIV-1. D’autre part, la structure nucléosomale du provirus intégré et les modifications épigénétiques associées jouent un rôle crucial pour l’expression des rétrovirus. La répression transcriptionnelle du rétrovirus oncogène BLV (Bovine Leukemia Virus) lui permet d’échapper au système immunitaire de son hôte bovin et favorise ainsi l’apparition de tumeurs. Dans ce contexte, nous avons montré que la méthylation de l’ADN au niveau du promoteur viral permet le maintient de la latence transcriptionnelle. En effet, la méthylation des dinucleotides CpGs localisés dans le LTR5‘ empêche le recrutement in vivo des facteurs de transcription activateurs CREB/CREM/ATF. Nous avons également montré que l’activation transcriptionnelle de l’expression du BLV par la combinaison PMA/ionomycine s’accompagne d’un remodelage chromatinien rapide mais transitoire au niveau du promoteur viral par des expériences de marquage indirect des extrémités et d’immunoprécipitation de chromatine. Nous avons ensuite démontré l’importance du site de liaison pour le facteur de transcription PU.1 et de la E-box 4 qui lie USF-1/-2, tous deux localisés dans la région dont l’accessibilité aux nucléases s’accroît après traitement des cellules, pour l’activation transcriptionnelle de l’expression virale par cette combinaison d’inducteurs. En conclusion, notre travail devrait permettre une meilleure compréhension des mécanismes transcriptionnels et épigénétiques régulant l’expression des rétrovirus HIV-1 et BLV.

Epigenetic studies hold the promise of addressing some of the fundamental questions of human biology including development, cell differentiation, and the aetiological mechanisms of complex disease. Over the last years, several new large scale high throughput technologies have been developed to allow genome wide profiling of epigenetic signals such as DNA methylation and histone modifications. Two of such technologies were developed in our laboratory enabling a genome wide microarray based profiling of DNA methylation signatures and a high throughput method for the site specific interrogation of the density of methylated cytosine. Using these techniques, we identified a DNA methylation difference in the 3’UTR of the DLX1 gene with potentially functional implications to discordance in risk taking behavior in a single pair of MZ twins. We modeled a power analysis on the effect size of the detected difference and determined that approximately 6~25 discordant twin pairs will be adequate to yield 80% power across the entire 12 K CpG island microarray platform using our epigenomic microarray profiling technique. We performed a DNA methylome analysis of MZ twins in white blood cells (WBC), buccal epithelial cells, and gut (rectum) biopsies (N=57 pairs in total) using 12K CpG island microarrays providing the basis for the first annotation of epigenetic metastability of ~6,000 unique genomic regions in MZ twins. We performed a classical twin study on DNA methylation differences in WBC and buccal epithelial cells from 39 pairs of MZ twins to 40 pairs of DZ twins. DZ co-twins exhibited significantly higher epigenetic difference compared to the MZ co-twins in buccal cells (p=1.2x10-294). While such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and comparison of methylomes in inbred vs. outbred mice favour the hypothesis that this is due to epigenomic differences in the zygotes. This study suggests that molecular mechanisms of heritability may not be limited to DNA sequence differences.

Epigenetic studies hold the promise of addressing some of the fundamental questions of human biology including development, cell differentiation, and the aetiological mechanisms of complex disease. Over the last years, several new large scale high throughput technologies have been developed to allow genome wide profiling of epigenetic signals such as DNA methylation and histone modifications. Two of such technologies were developed in our laboratory enabling a genome wide microarray based profiling of DNA methylation signatures and a high throughput method for the site specific interrogation of the density of methylated cytosine. Using these techniques, we identified a DNA methylation difference in the 3’UTR of the DLX1 gene with potentially functional implications to discordance in risk taking behavior in a single pair of MZ twins. We modeled a power analysis on the effect size of the detected difference and determined that approximately 6~25 discordant twin pairs will be adequate to yield 80% power across the entire 12 K CpG island microarray platform using our epigenomic microarray profiling technique. We performed a DNA methylome analysis of MZ twins in white blood cells (WBC), buccal epithelial cells, and gut (rectum) biopsies (N=57 pairs in total) using 12K CpG island microarrays providing the basis for the first annotation of epigenetic metastability of ~6,000 unique genomic regions in MZ twins. We performed a classical twin study on DNA methylation differences in WBC and buccal epithelial cells from 39 pairs of MZ twins to 40 pairs of DZ twins. DZ co-twins exhibited significantly higher epigenetic difference compared to the MZ co-twins in buccal cells (p=1.2x10-294). While such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and comparison of methylomes in inbred vs. outbred mice favour the hypothesis that this is due to epigenomic differences in the zygotes. This study suggests that molecular mechanisms of heritability may not be limited to DNA sequence differences.

Mechanisms of arsenic toxicity are not yet clear. Arsenite has effects on methylation pathways, by decreasing expression of DNA methylases and depletion of S-adenosylmethionine. Histones are DNA packing proteins that regulate gene expression modulating chromatin accessibility. Methylation at Lysine 9 of Histone H3 (K9H3) is a hallmark of heterochromatin. Dimethyl K9H3 is a mark of facultative heterochromatin and trimethyl K9H3 is present on constitutive heterochromatin. HeLa cells exposed for 24 hrs to 1 uM or 5 uM Sodium Arsenite were fixed and different posttranslational modifications of histones were detected by indirect immunofluorescence. Images were analyzed to assess the change on average methylated species of K9H3 in cell nuclei. Interestingly Arsenite (1 uM and 5 uM) treated cells had a significant increase in the trimethylated and dimethylated of K9H3, evaluated throught the comparison of average nuclei brightness and pixel value analysis between treatments.

Apomixis is an intriguing and agronomically valuable asexual reproductive pathway resulting in seeds that give rise to plants that are identical in genotype to the female parent. Apomixis is absent in agriculturally important seed crops and our work has focused on the analysis of apomixis in the daisy-like genus Hieracium which contains sexual and apomictic species. Prior studies have shown that apomixis in Hieracium is controlled by two dominant loci. The LOA controls the avoidance of meiosis during female gametophyte (embryo sac) formation and the LOP locus is required for fertilization independent embryo formation during seed initiation. The genes conferring apomixis are unknown. In this study we focused on the events of autonomous seed initiation. Cytological examination of apomictic mutants that have lost LOA or LOP and analysis of their progeny enabled us to characterize developmental aspects associated with the function of these loci. Upon removal of LOA meiosis occurs normally and LOP segregates with a 1:1 ratio in the progeny, characterizing maternal gametophytic control. We also show that autonomous embryo formation segregates with autonomous endosperm formation, suggesting that these two loci are closely linked. However, upon meiotic division, embryo lethal components arise and embryo development in apomeiosis mutants was generally defective and seed set was low. Similarly, upon removal of LOP, apomixis initiation occurs normally and unreduced embryo sacs can only form seeds if pollinated. Autonomous seed initiation is actively repressed in the sexual model plant Arabidopsis by the action of a chromatin remodelling complex encoded by the FERTILIZATION INDEPENDENT SEED (FIS)-class genes. These genes are homologues of the Drosophila PcG complex that also repress gene expression throughout Drosophila development. Mutations in the FIS-class genes lead to elements of apomixis, such as autonomous endosperm, and in one particular mutant, autonomous egg cell development. Given the similarity in apomictic and FISclass gene mutant phenotype we isolated three homologues from sexual and apomictic Hieracium plants: FERTILIZATION INDEPENDENT ENDOSPERM (FIE), MULTICOPY SUPPRESSOR OF IRA1 (MSI1) AND RETINOBLASTOMA (RBR). FIS-class genes from sexual and apomicit Hieracium and examined their expression, interaction and function during seed initiation. The isolated Hieracium FIS-class genes were highly conserved in sexual and apomictic plants in terms of gene sequence and temporal and spatial expression pattern. Analysis of protein interactions by yeast-two hybrid showed that the HFIE gene from sexual and apomictic plants does not interact with other complex members in the same manner found in Arabidopsis. Protein modelling uncovered structural differences between the Arabidopsis and Hieracium FIE proteins. RNAi- mediated down-regulation of HFIE in sexual Hieracium did not lead to autonomous seed initiation indicating HFIE was not part of a repressive complex. Down-regulation of HFIE in sexual and apomictic plants revealed the gene was essential for embryo growth and viability. Therefore, FIS-complex genes interact differently in Arabidopsis and Hieracium and have different developmental roles. In summary, the results presented here suggest that the FIS-genes are not mutated in apomictic Hieracium plants, but they interact differently relative to the Arabidopsis counterparts and play a fundamental role in embryogenesis. Thus, engineering autonomous seed into crops will not depend on mutating these genes but rather in uncovering the molecular signal that triggers apomictic development. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337073 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19613号 / 医博第4120号 / 新制||医||1015(附属図書館) / 32649 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 岩田 想, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Developing oilseeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated at the transcriptional level through the transcription factors belonging to the LAFL regulatory network. The VAL genes encode repressors of the seed maturation program in germinating seeds, but they are also expressed during early stages of seed maturation. VAL1 was identified through a reverse genetics approach as a regulator of seed metabolism, as val1 mutant seeds accumulated elevated levels of storage proteins compared to the wild type. Two VAL1 splice variants were identified, yielding the canonical protein and a truncated protein lacking the plant-homeodomain-like domain important for epigenetic repression. Transcriptomics analysis also revealed that VAL1 is a global epigenetic and transcriptional repressor in developing embryos, though none of the transcripts encoding the LAFL network regulators, including FUSCA3, were affected in val1 embryos. However, VAL1 action is connected specifically to FUSCA3 as 38% of transcripts belonging to the FUSCA3 regulon, but not to other regulons, were largely de-repressed in the absence of VAL1. Based on our model, FUSCA3 activates expression of VAL1 to repress transcription of seed maturation genes without interfering with expression of the core LAFL regulators. / Ph. D.

P53 empêche la réplication d'ADN cellulaire pathologique ainsi que viral en activant la transcription d'effecteurs en aval. L'augmentation d'expression de p53 et sa phosphorylation en réponse à des oncogènes ou à des dommages à l'ADN sont considérées comme déterminant l'activation transcriptionnelle de p53. Les mutations tumorales et les protéines virales convergent fonctionnellement en inactivant p53. Par exemple la protéine cellulaire MDM2 et la protéine adénovirale E1 B-55K ciblent toutes les deux p53 pour sa dégradation. Ceci représente la base du raisonnement de la création des molécules antagonistes de MDM2 et de l'adénovirus oncolytique où E1B-55K a été supprimé, ONYX-015 (Oncorine), comme thérapies anticancéreuses ciblant p53. Pourtant ici, nous montrons que E1B-55K est dispensable pour l'inactivation de p53 et nous révélons un mécanisme épigénétique nouveau et dominant qui inactive l'activité de p53, quel que soit le niveau et la phosphorylation de p53. En utilisant une approche génétique, nous dévoilons qu'E4-ORF3, une autre protéine adénovirale, est le point de nucléation de domaines d'hétérochromatine, menant à l'inactivation des promoteurs cibles de p53, en empêchant sa liaison et donc son activation transcriptionnelle. De plus, nous montrons qu'E4-ORF3 forme un échafaudage nucléaire qui dirige deux métyltransferases, SUV39H1 et SUV39H2, vers ces nouveaux domaines répressifs. Notre étude change la définition fondamentale du mécanisme d'inactivation de p53 dans les cellules infectées par un adénovirus, apportant une nouvelle lumière sur un mécanisme critique qui va maintenant permettre le développement de thérapies adénovirale vraiment sélectives du statut p53 des cellules. / P53 guards against pathological cellular and viral DNA replication by activating the transcription of downstream effectors. The induction of p53 levels and its phosphorylation in response to oncogenes and DNA damage is thought to determine p53 transcriptional activation. Tumor mutations and viral proteins functionally converge in inactivating p53. For example the cellular protein MDM2 and the adenoviral protein E1B-55k both target p53 for its degradation. This is the premise for MDM2 antagonists and the E1B-55k deleted oncolytic adenovirus, ONYX-015 (Oncorine) as p53-targeted cancer therapies. However, here we show that E1B-55k is dispensable for p53 inactivation and reveal a novel and dominant epigenetic mechanism that silences p53 activity, irrespective of p53 level and phosphorylation. Using a genetic approach, we reveal that E4-ORF3, another adenoviral protein, nucleates heterochromatin domain, leading to the silencing of p53 target promot er by preventing its binding and subsequent transcriptional activation. Moreover we show that E4-ORF3 forms a novel nuclear scaffold that directs two methyltransferases, SUV39H1 and SUV39H2, to this newly formed repressive domains. Our study changes the fundamental definition of how p53 is inactivated in adenovirus infected cells, and provides a critical mechanistic insight that could now enable the rational development of true p53 tumor selective adenoviral therapies.

Non-melanoma skin (NMSC) cancer is the most common human malignancy. Cutaneous squamous cell carcinoma (cSCC) and its precursor, actinic keratosis (AK) affect tens of thousands of people each year in the UK. Merkel cell carcinoma is a rare, yet aggressive type of NMSC recently linked with Merkel Cell Polyomavirus (MCPyV). In spite of the clinical burden of NMSC, key molecular regulatory patterns remain largely unknown. The aims of this thesis were to investigate genome-wide genetic, epigenetic and transcriptional changes in AK and cSCC, and assess the prevalence of MCPyV and its effect on methylation in NMSC. Copy-number analysis revealed that AK harbours significantly more genomic aberrations compared to skin, the majority of which occurs on chromosomes 8 and 9. Transcriptional profiling has found 292 and 308 genes as differentially expressed in AK compared to non-sunexposed and sun-exposed skin, respectively, and gene-set enrichment analysis (GSEA) revealed dysregulation of PPAR pathway in this lesion. Expression profiling of cSCC and AK has revealed 346 differentially expressed genes, and GSEA detected dysregulation in several canonical pathways including TGF-β and MAPK pathway. Aberrant methylation in cSCC cell lines occurs in the promoters of many developmental genes. A total of 1085 hyper- and 833 hypomethylated genes were detected in cSCCs, and GSEA revealed dysregulation of critical signalling pathways (WNT, MAPK signalling pathways). Methylation analysis of AK revealed a total of 4194 differentially methylated genes, and implicated FOXF2, PITX2, RUNX1 and SMAD3 transcription factors in this lesions. MiRNA profiling of cSCC and normal skin revealed significant dysregulation of 38 miRNAs including several of viral origin. MCPyV was shown to be common in NMSC, yet MCPyV nor human papillomavirus does not affect cSCC methylation. Taken together, this work provides novel insight into molecular regulation of cSCC oncogenesis, and identifies potential epigenetic targets for functional evaluation in this malignancy.