Background. VAV interacting Krüppel-like factor (VIK) is a novel transcription factor. Previously our lab reported a series of breast cancer tumour samples where VIK methylation was associated with an increased risk of recurrence in tamoxifen-treated patients, indicating a role for VIK in ER positive breast cancer and endocrine resistance. Additionally VIK has been shown to be involved in cell cycle regulation, interacting with CDK4 and VAV1. The cyclin D-CDK4/6-Rb pathway is frequently dysregulated in ER positive breast cancer. Combined treatment of palbociclib, a highly selective CDK4/6 inhibitor, with endocrine therapy substantially improved outcome of patients with ER positive metastatic breast cancer. Increasing clinical use means acquired resistance to palbociclib is emerging as a major clinical challenge. Results. VIK was confirmed to be subject to regulation by DNA methylation in breast cancer. VIK methylation correlated to transcriptional silencing of mRNA in both cancer cell lines and primary tumours. To determine the functional significance of loss of VIK expression, VIK was knocked down in unmethylated breast cancer cell lines and a normal breast epithelial cell line. Knockdown resulted in cell death via induction of apoptosis. VIK knockdown altered cell cycle progression from G1 to S phase. Expression of multiple regulatory cell cycle proteins was altered, differentially in normal and tumour cells. Treatment with the CDK4/6 inhibitor, palbociclib, in cells with reduced VIK expression resulted in decreased sensitivity to the drug, inducing a shift in IC50 value towards resistance. In a model of acquired resistance, T47D cells were cultured long-term with palbociclib generating resistant clones. VIK was significantly downregulated in all resistant clones to barely detectable mRNA levels, suggesting a role for VIK in resistance to CDK4/6 inhibition. Conclusion. This PhD has confirmed VIK is a novel epigenetically regulated gene in breast cancer. VIK expression is essential to both normal and tumour breast cell survival and is involved in the regulation of the G1/S phase transition in the cell cycle. Loss of VIK expression potentially contributes to the development of acquired resistance to CDK4/6 inhibitors.

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.

Entomopathogenic fungi, including Beauveria bassiana are being developed as an alternative to chemical insecticides. Their effectiveness can be enhanced through understanding of the mechanisms of response to environmental stresses and conditions. An aspect of repair of ultraviolet radiation induced DNA damage and response to high temperature were studied here. A region of the photolyase gene (phr-1), encoding cyclobutane pyrimidine dimer photolyase (CPD-PHR), an enzyme pivotal to DNA repair, was cloned, sequenced and identified for species of the genera Beauveria, Isaria, Lecanicillium, Metarhizium and Tolypocladium. The DNA and deduced amino acid sequences were analysed using several in silico methods and annotated for functionality. The data suggested that the DNA encoded a protein with conserved residues known in CPD-PHR function, which had structural homology with other CPD-PHRs and molecular phylogeny that was generally consistent amongst this group of fungi. These results are the first for a phr-1 from the genera Isaria, Lecanicillium and Tolypocladium. In bacteria and yeasts, tolerance to environmental stress was shown to be aided through an inducible phenomenon that involves extracellular sensory component (ESC) proteins in Escherichia coli, which have yet to be purified or sequenced. The presence of an ESC-like factor (ELF) was examined in cell-free filtrate (CFF) from B. bassiana cultures. It was revealed that the tolerance of conidiospores and blastospores (BS) to ultraviolet radiation or heat could be increased by preheated CFF, respectively, but not after pretreating the CFF with trypsin. Several novel polyacrylamide-based in situ and binding bioassays were developed to screen for and characterize ELF candidate (EC) proteins. Two were detected (EC1 and EC2) and EC1 was found to interact with BS, while bioassays with purified ECs showed that EC1 could increase heat tolerance in BS. De novo peptide sequencing revealed that the ECs were the same protein, but differed by EC1 being glycosylated. An expressed sequence tag from B. bassiana, encoding six peptides that were also found in the ECs, was identified in the public data base. This sequence information was exploited to amplify the remaining coding regions of the suspected ELF gene (elf) using polymerase chain reactions. Through this a 741 nucleotide open reading frame was cloned and sequenced. Structure-function analyses of the amino acid sequence encoded by the open reading frame revealed features that were consistent with the ECs, such as eight shared peptides, its nascent derived size (26 kDa), potential glycosylation sites and secretion signal peptide. In addition, other features such as the high proportion of cysteine residues and internal amino acid repeats will be discussed. The elf gene was inserted into an expression vector and introduced into the methylotrophic yeast Pichia pastoris for its controlled over-expression. Heterologously expressed ELF conferred elevated tolerance to heat in BS to similar levels produced by ELF synthesized by B. bassiana. Several functional and molecular features of the ELF system have certain commonalities with many agonist-receptor systems involved in signal transduction, but remain to be detailed. This is the first report of the cloning and functional analyses of elf and ELF, respectively, from any organism.

Krüppel-like factors (KLFs) are a family of 3 Cys2/His2 zinc finger transcription factors with a diverse set of roles in cellular differentiation, cell cycle regulation, tumor suppression, erythropoiesis, angiogenesis, and other processes. During embryonic development, KLF2 has a role in vessel maturation. Adult conditional KLF4 knockout mouse embryos have thickened arterial intima follow vascular injury. Breeding KLF2+/- and KLF4+/- mice resulted in the generation of KLF2/KLF4 double knockout (DKO) embryos. KLF2/KLF4 DKO embryos died by E10.5 with cranial bleeding. Using immunohistochemistry, embryo whole-mounts were examined for differences in gross vascularization between wild-type (WT), KLF2-/- and KLF2/KLF4 (DKO embryonic day 9.5 (E9.5) embryos. No obvious gross capillary abnormalities were noted in E9.5 KLF2/KLF4 DKOs, although the posterior cardinal vein appeared to narrow rostral to caudal in KLF2-/- and KLF2/KLF4 DKO embryos. Light and electronic microscopy were employed to investigate potential structural and ultrastructural phenotypes in KLF2/KLF4 DKO embryos. Microscopy confirmed hemorrhaging near and endothelial breaks in the primary head vein (PHV) in E9.5 KLF2/KLF4 DKOs (n=8) and E10.5 KLF2-/-KLF4+/- embryos (n=1). Electron micrographs illustrated a disrupted endothelium in KLF2/KLF4 DKOs with endothelial cells having filopodia-like projections. Surprisingly, KLF2-/- embryos had the presence of wider medial PHV endothelial gaps compared to WT at the electron micrograph level. Density counts revealed a 15% reduction in midline cranial mesenchyme at the level of hemorrhaging in KLF2/KLF4 DKOs compared to KLF2-/- (n=3). An in-situ hybridization localized KLF2 RNA expression to the endothelium of the PHV. A quantitative reverse transcriptase polymerase chain reaction assay revealed that the eNOS expression is synergistically regulated by KLF2 and KLF4, as a shared downstream target. It is proposed that KLF2 and KLF4 share in the regulation of multiple gene targets, leading to early death by E10.5.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

Relaxin-like factor (RLF), also known as Leydig insulin-like peptide (Ley-I-L) or Insulin 3 (INSL3), is a newly characterized member of the insulin peptide family. Amino acid sequence homology revealed that RLF is more closely related to relaxin than any other insulin-like hormones. The main aim of this thesis was to sequence the rat RLF (Relaxin-like factor) gene and determine the structure and organisation of the gene. Secondly to compare the structural organisation of the rat RLF/JAK3 genomic region with that of the mouse and human, using bioinformatic databases. Thirdly to further investigate the signalling pathways for the RLF receptor, in particular the NFƒÛB pathway. The homology between rat and mouse in the JAK3/RLF region revealed 84.4 % similarity over 1262 bp of DNA sequence, observing that unlike the mouse, the rat RLF promoter is separated from the JAK3 gene by around 700-1000 bp. Similarly in humans, the RLF gene is located around 4 kb downstre am from JAK3. Also Protein kinase A (PKA) was the only signalling pathway which dispalyed major induction and no inhibitory effects were observed through the NFƒÛB signalling pathway.

<p>The progesterone receptor (PR) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. The steroid hormone progesterone binds to PR and induces a conformational change that enables the receptor to bind DNA, recruit cofactors, and directly regulate the transcription of target genes. In addition, extra-nuclear PR can indirectly regulate gene expression by rapidly activating other signaling pathways such as Src/MAPK. Although the direct and indirect functions of PR have been well studied in isolation, it is important to understand the molecular mechanisms by which these pathways can cross talk and integrate to ultimately impact gene expression.</p><p>Towards this end, we initiated studies to assess the overall impact of MAPK inhibition on PR transcriptional activity in T47D breast cancer cells treated with the synthetic progestin R5020. During the course of microarray and biochemical analyses that were undertaken to address this issue, we discovered a subset of PR target genes that are enriched for E2F binding sites. Subsequently, we determined that PR-B is a component of several distinct pathways that function both directly and indirectly to positively up-regulate E2F1 expression in T47D breast cancer cells. Firstly, PR directly regulates E2F1 transcription by binding to proximal and distal enhancer sites located near E2F1. Secondly, progestin induces the hyperphosphorylation of Rb, which results in increased recruitment of E2F1 to its own promoter, thereby activating a positive feedback loop that further amplifies its transcription. Finally, PR induces expression of Krüppel-like factor 15 (KLF15) and potentially other Sp/KLF family members, which can bind to GC-rich DNA within the E2F1 promoter and further activate transcription. Together, these results suggest a paradigm for multimodal PR gene regulation that entails cooperation between direct and indirect pathways of PR signaling to achieve the desired downstream transcriptional cascade.</p><p>In the breast and other tissues of the female reproductive system, progesterone plays an important role in normal development and function. Therefore, synthetic PR modulators (PRMs) are widely used to manipulate the downstream biology of PR for purposes including contraception and hormone replacement therapy (HRT). However, progestins and PR have also been implicated in disease pathologies such as breast cancer. While the molecular mechanisms by which PR regulates breast tumor growth have not been fully elucidated, recent studies highlight the fact that progestins may have a dose-dependent role in breast cancer progression. Consequently, we undertook studies to identify and characterize any differential effects of low-dose versus high-dose progestins on the downstream activities of PR. Specifically, we found that treatment of breast cancer cells with low-dose progestins can induce maximal transcriptional activation of a subset of PR target genes, including the cell cycle regulators cyclin D1 and E2F1. Furthermore, low-dose and high-dose progestins have differential effects on the phosphorylation of PR and subsequent receptor turnover. Cumulatively, these findings underscore the importance of establishing the effects of a wide range of progestin concentrations on target gene expression and other PR actions, so that we are able to accurately predict the potential consequences of PRMs on downstream PR signaling pathways and biology.</p> / Dissertation

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.