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Cytosine methylation of CpG dinucleotides is an epigenetic modification that cells use to regulate gene expression, largely to promote transcriptional silencing. Focal hypermethylation of tumor suppressor genes (TSGs) accompanied by genomic hypomethylation are epigenetic hallmarks of malignancy. DNA methyltransferase 1 (DNMT1) is the principle vertebrate enzyme responsible for maintenance of DNA methylation and its dysregulation has been found to lead to aberrant methylation in cancer. In addition, recent findings demonstrated that the ten-eleven translocation 1 (TET1) protein functions as a 5-methylcytosine dioxygenase that converts 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC) to mediate active DNA demethylation. Emerging evidence suggests that TET1 might function as a TSG. To understand the dynamic regulation of DNA methylation during cellular transformation, my work focused on intrinsic regulation of DNMT1 and how TET1 regulates DNA demethylation in generating a cancer methylome. The replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, other data suggested that RFTS may be a positively acting domain. To test biochemical and structural predictions that the RFTS domain of DNMT1 is inhibitory, we established cellular systems to evaluate the function of DNMT1 alleles. The data indicate that deletion of RFTS is necessary and sufficient to promote cellular transformation, focal hypermethylation of specific TSGs, and global hypomethylation. These data and human mutation data suggest that RFTS domain is a target of tumor-specific dysregulation. RAS mutations are frequently in multiple malignancies. Methylation-associated silencing of TSGs is a hallmark of RAS-driven-tumorigenesis. I discovered that suppression of TET1 by the ERK signaling cascade is responsible for promoter hypermethylation and the malignant phenotype in KRAS-transformed cells. Restoration of TET1 expression reactivates silenced TSGs and reduces colony formation. Moreover, TET1 knockdown in a cell depleted for KRAS is sufficient to rescue the inhibition of colony formation by KRAS knockdown. My findings suggest that dysregulated TET1-mediated DNA demethylation is a target responsible for epigenetic changes in cancers with KRAS activation.

Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.

Résumé Parmi les rétrovirus tumorigènes, les rétrovirus appelés « complexes » dont font partie HTLV-1 chez l’homme et BLV chez le mouton, sont responsables de leucémies lymphoïdes chroniques caractérisées par des pathogenèses similaires. Bien que la contribution essentielle de la protéine Tax dans la leucémogenèse soit bien établie pour ces deux virus, il existe toujours une contreverse quant à l’expression des protéines virales -dont l’oncoprotéine Tax- dans les cellules transformées. Dans le cas de HTLV-1 et BLV, le virus est latent. Une hypothèse attrayante suggère que l’extinction complète du provirus accompagnée du blocage transcriptionnel de l’expression de Tax dans la cellule leucémique serait un élément indispensable au développement de la tumeur en réduisant son immunogénicité. Le silencing viral permettrait à la cellule infectée d’échapper à la reconnaissance par le système immunitaire de l’hôte. Dans la première partie de notre travail, nous avons pu montrer, grâce à l’observation et le suivi de deux moutons infectés par BLV, que l’extinction virale était associée au développement tumoral. Alors que la phase asymptomatique est caractérisée par l’existence de différents clones cellulaires infectés et exprimant le virus, la phase tumorale est caractérisée par l’existence d’un seul clone cellulaire dans lequel l’expression virale est éteinte. Dans le cas du mouton S2531, nous avons mis en évidence que le silencing observé dans la phase tumorale est d’origine génétique. L’extinction de l’expression virale et la domination du clone muté au niveau de la protéine Tax (K303) sont associées à l’émergence de la leucémie et constituent une caractéristique de la phase tumorale. Le deuxième mouton étudié, S267, est caractérisé par la présence de cellules infectées non transformées et transformées au même moment. Par ailleurs, le mouton S267 est caractérisé par une absence de mutations ou délétions au niveau du provirus intégré dans les cellules tumorales. Dans la deuxième partie de notre travail, nous avons pu, grâce à l’établissement de la lignée L267 dans laquelle le silencing viral n’est pas lié à une défection dans la structure génomique du provirus, élucider les mécanismes épigénétiques responsables du silencing. Nous montrons que ce provirus silencieux peut être réactivé in vitro 1) lorsque la protéine Tax sauvage est introduite par transfert rétroviral, 2) après traitement des cellules par la trichostatine A (TSA), un inhibiteur des histones désacétylases, 3) par traitement des cellules à la 5’azacytidine, un agent inhibiteur de DNA méthyltransférases. La réactivation du provirus silencieux lui confère la capacité d’infecter des moutons sains, suggérant que le provirus est complet et exempt de mutation qui pourrait altérer son fonctionnement. Les complexes de désacétylation des histones msin3A et HDAC-1 jouent un rôle important dans l’établissement du silencing viral via la condensation de la chromatine, et ce changement de la structure chromatinienne est lié a un ensemble de modifications ciblant les acides aminés des queues N-terminales des histones H3 et H4 établissant ainsi un code histone pour l’extinction et l’expression du provirus. Enfin, l’étude comparative des profils d’expression génique de cellules B tumorales dans lesquelles le provirus BLV est soit silencieux, soit ré-activé suite à l’expression exogène de Tax a montré que les mécanismes épigénétiques de silencing se superposent même dans le modèle génétique de silencing. De plus le rétablissement de l’expression virale par Tax est associé à une élimination des complexes de désacétylation des histones au niveau du promoteur viral. Nos résultats ouvrent le champ vers l’utilisation de modèle de leucémie ovine dans des essais thérapeutiques basés sur la combinaison du traitement par des inhibiteurs des HDACs et des DNMTs et une immunothérapie ciblant des antigènes tumoraux d’origine virale ou cellulaire. De telles expériences in vivo constitueront un modèle utile pour le traitement l’ATLL et de pathologies prolifératives touchant les cellules B chez l’homme.

Cancer is a leading cause of death worldwide, accounting for around 13% of all death [1]. Traditional cancer therapeutics usually require careful selection of one or more intervention, such as surgery, radiotherapy, and chemotherapy, which have made momentous progress, but have ample limitations [2]. The next generation of cancer therapeutics will specifically target processes responsible for the growth and survival of cancer cells. Among the most promising of these molecularly-targeted therapeutics are short interfering RNAs (siRNAs). These siRNAs serve as the effectors of RNA interference, a naturally occurring and highly specific mechanism for regulating gene expression through sequence-specific degradation of messenger RNA. However, the native structure of RNA is plagued with undesirable chemical properties. For example, the sugar-phosphate backbone contains a negative charge which hinders its ability to cross the negatively charged lipid bilayer. Furthermore, the phosphodiester backbone is a substrate for nucleases, which catalytically cleaves the phosphate-oxygen bond, thus degrading the native RNA [3]. As such, there is widespread interest in chemically modifying the backbone of siRNAs in order to overcome some of the inherent problems with its native structure. There have been only two reports that have employed amide-bond linkages as phosphate replacements within siRNAs [4, 5]. In both of these studies, the amide bond containing monomer units were placed at the 3’-overhangs and not within the internal Watson-Crick region of the double stranded siRNA due to the limitation of standard solid-phase oligonucleotide synthesis. In this thesis, we proposed to utilize phosphoramidite chemistry to localize internal amide-bond modifications [6]. A practical synthesis of a peptide nucleic acid unit combined with an RNA nucleoside (PNA-RNA dimer, UaU) is reported [7]. Using this PNA-RNA dimer phosphoramidite allows us to control the site-specific location of the internal amide-bond modification throughout the desired RNA strand. Polyacrylamide gel (PAGE) and mass spectrometry analysis were performed to ensure the formation of full-length modified siRNA molecules. The effects of these modifications were explored with respect to the biophysical and biological properties of the modified siRNAs. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), cell-based luciferase assays, and nuclease stability assays. Melting temperature experiment reveals that localizing a UaU dimer unit within the RNA oligonucleotides has an overall destabilizing effect, whereas UaU modifications at the 3’-overhang positions show little change in thermal stability. Circular dichroism experimental results illustrate that all chemically modified siRNAs exhibit the standard A-form helix. In cell-based luciferase assays, we utilized two different target sequences and our results highlight the compatibility of utilizing a neutral amide-bond backbone within siRNAs. Specifically, the internal amide-bond modification is compatible within the RNAi machinery when placed at 3’-overhang position in the sense strand of the double-stranded siRNA. However, poor efficacy is observed when this unit is placed adjacent the Ago 2 cleavage site on the antisense strand. The nuclease stability assays reveal that the introduction of a PNA-RNA dimer at the 3’-end of the siRNA where the exonuclease cleaves the terminal nucleotide, increased markedly the resistance to serum-derived nucleases. To the best of our knowledge, this is the first report that involves amide-bonds as phosphate backbone replacements within the internal regions of siRNAs and thus opens the future possibility for examining and utilizing this modification in studying new structure-function relationships. / UOIT

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.

The presence of an unpaired copy of a gene during meiosis triggers the silencing of every copy of that gene in the diploid ascus cell of Neurospora crassa, a phenomenon called Meiotic Silencing. This phenomenon has two stages: trans-sensing and meiotic silencing. If a DNA region is not detected on the opposite homologous chromosome early in meiosis (a trans-sensing failure), a signal corresponding to the unpaired region is produced that transiently silences expression of all homologous sequences. Meiotic silencing is related to RNA Silencing, a phenomenon that employs RNA-dependent RNA Polymerases (RdRPs), Argonautes, and Dicers. Dicers cleave double-stranded RNA (dsRNA) into 21-23 nucleotide RNAs. In the filamentous fungus Neurospora crassa, two RNA silencing pathways have been identified; one is active during mitosis, and the other is active during meiosis. The mitotic RNA silencing pathway, known as “quelling”, involves an RdRP (quelling-deficient-1--qde-1), an Argonaute-like protein (quelling-deficient-2--qde-2), and two Dicer-like proteins (dicer-like-1--dcl-1 and dicer-like-2--dcl-2). Previous studies in N. crassa also revealed the involvement of an RdRP (Suppressor of ascus dominance-1--Sad-1) and an Argonaute-like protein (Suppressor of meiotic silencing-2--Sms-2) in meiotic silencing, suggesting that meiotic silencing is RNA-dependent and raising the question of whether a Dicer is involved in meiotic silencing. In this work, we tested the participation in meiotic silencing of the dcl-1 gene of N. crassa, which codes for a Dicer-like protein we call Suppressor of meiotic silencing-3--Sms-3. Crosses homozygous for mutant alleles of Sms-3 are barren, indicating that the gene is also essential for sexual development. Due to this homozygous sterility, we could only test the involvement of Sms-3 in meiotic silencing in heterozygous crosses. Under these conditions, we observed suppression of the meiotic silencing which would have otherwise been induced by the presence of unpaired DNA of reporter genes. We conclude that the Dicerlike protein Sms-3 is required for both meiotic RNA silencing and sexual development.

In S. cerevisiae, the ribosomal DNA locus is silent for RNA polymerase II (Pol II) transcription and recombination (rDNA silencing). Our goal is to understand how histones and histone-modifying enzymes regulate the silent chromatin at the rDNA locus. Sir2, a NAD+-dependent histone deacetylase, is required for rDNA silencing. To understand how Sir2 regulates rDNA silencing, we performed chromatin immunoprecipitation to measure the association of modified histones across the rDNA repeat in wild-type and sir2Δ cells. We found that in sir2Δ cells, histone H3 at the rDNA became hyperacetylated and hypermethylated. High levels of K4-methylated H3 correlate with Pol II transcription. Consistent with this, we found that the nontranscribed spacer (NTS) region was transcribed by Pol II in sir2Δ cells. To investigate if transcription of the NTS region regulates rDNA silencing, we overexpressed this region both in trans and in cis. Our data showed that overexpression of the NTS region in cis caused Pol II silencing defect and hyperrecombination at the rDNA. These data suggest that Sir2 contributes to maintain the silent chromatin at the rDNA by repressing Pol II transcription in the NTS region. We also found that the NTS transcripts could be translated in vitro and that they copurified with polysomes, suggesting that the transcripts may encode proteins or that the transcripts are somehow involved in the process of translation. Additionally, we examined the role of linker histone H1 in regulating rDNA silencing. We found that, unlike Sir2 that represses both Pol II transcription and recombination, histone H1 only represses recombination at the rDNA. The hyperrecombination defect at the rDNA is more severe in sir2Δ hho1Δ double mutant than in either single mutant, suggesting histone H1 and Sir2 act independently. Consistently, hho1Δ cells did not accumulate extrachromosomal rDNA circles (ERCs) or the Holliday junction intermediates, which accumulate in sir2Δ cells. These data suggest that histone H1 and Sir2 regulate different recombination pathways. In summary, my research has provided insight into the mechanism of how silent chromatin at the rDNA locus is regulated, which will help us understand how fundamental components of chromosomes affect gene expression and genome stability.

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