<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Both microRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate post- transcriptional events, but the post-transcriptional contribution to the global mammalian transcriptomes is still not well understood. In this study we study the synergistic interaction between microRNAs that inhibit gene production, and a special RBP, HuR, that positively regulates mRNA stability. We examined their relationship in terms of spatial, conservational and expressional perspective. We show comprehensive mapping of HuR binding sites by combination of its structural and sequential preferences; and cross-platform normalization method within a process of refining miRNA and HuR binding site mapping. Finally, we observed co-evolution of miRNA and HuR binding sites by looking at their proximity and conservation levels. Collectively, our data suggest that mammalian microRNAs and HuR, with seemingly opposing regulatory effects, cooperatively regulate their mutual targets.

Gene expression can be regulated at multiple steps once transcription is initiated. I have studied two different gene models, the α-Fetoprotein (AFP) and the immunoglobulin heavy chain (IgM) genes, to better understand post-transcriptional gene regulation mechanisms. The AFP gene is highly expressed during fetal liver development and dramatically repressed after birth. There is a mouse strain-specific difference between adult levels of AFP, with BALB/cJ mice expressing 10 to 20-fold higher levels compared to other mouse strains. BALB/cJ mice express low levels of Zhx2 and thus incompletely repress AFP. Despite differences in steady state AFP mRNA levels in the adult liver between Balb/cJ and wild-type mice, transcription rates across this gene were similar, indicating a post-transcriptional regulatory mechanism. I found accumulated unspliced RNA across multiple AFP introns in wild-type mice where mature AFP mRNA levels are low, suggesting overall AFP splicing is inefficient in the presence of Zhx2. The IgM gene is alternative processed to produce two mRNA isoforms through a competition between cleavage/polyadenylation (μspA) and splicing reactions and the pA/splice RNA expression ratio increases during B cell maturation. Cotranscriptional cleavage (CoTC) events, driven by specific cis-acting elements, are required downstream of some poly(A) signals to terminate transcription. In some cases, a pause site can produce similar effect. I explored whether there is a CoTC-like element within the IgM gene that may contribute to developmental changes in the mRNA ratio. In both a B cell and plasma cell line there was a gradual decrease in transcripts downstream from the μspA signal, suggesting that there is not evidence for a CoTC element within the IgM gene. To examine the effect a CoTC element would have on the competition between the splice and μspA reactions, we inserted the CoTC sequence of the β-globin gene into different locations downstream of the μspA signal. While the β-globin CoTC element caused cotranscriptional cleavage in all locations, it only affected the μspA/splice ratio when located close to the μspA site. This suggests there is a position effect of the inserted CoTC element on the competing polyadenylation and splicing reactions within the IgM transcripts.

AGAMOUS-Like 15 (AGL15) encodes a MADS-domain transcription factor that is preferentially expressed in the plant embryo, and may function as a regulator in embryonic developmental programs. A number of direct downstream targets of AGL15 have been identified, and while some of these target genes are induced in response to AGL15, others are repressed. Additionally, direct target genes have been analyzed that exhibit strong association with AGL15 in vivo, yet in vitro, AGL15 binds only weakly. Taken together these data suggest that AGL15 may form heterodimers, or ternary complexes with other proteins, thus modulating the specificity and function of AGL15 in planta. Yeast two-hybrid screens were undertaken to identify novel proteins able to interact with AGL15, and a number of interesting and potentially biologically important AGL15-interacting partners are reported here. These include members of a histone deacetylase complex, a COLD SHOCK DOMAIN (CSD)-containing protein, a Khomology domain/CCCH type zinc finger containing protein, a bZIP transcription factor, a homeobox-leucine zipper protein, a LATERAL ORGAN BOUNDARIES (LOB) domain containing protein, and an Agenet domain containing protein. Interactions between AGL15 and other MADS domain factors that are expressed in embryonic tissue, including SEPALLATA 3 (SEP3) have also been indentified. The regions of AGL15 that mediate interactions with the aforementioned proteins were mapped, and the capacity of these proteins to interact with other plant MADS-domain proteins tested. It is reported herein that AGL15 interacts with members of the SWI-INDEPENDENT 3/HISTONE DEACETYLASE (SIN3/HDAC) complex, and that AGL15 target genes are also responsive to an AGL15 interacting protein that is also a member of this complex, SIN3 ASSOCIATED POLYPEPTIDE OF 18 KD (SAP18). AGL15 can repress transcription in vivo, and a region essential to this repressive function contains an LxLxL motif that is conserved among putative orthologs of AGL15. What is more, the aforementioned motif mediates the association of AGL15 with SAP18 in yeast two-hybrid assays, thus providing a possible mechanism for explaining how role AGL15 regulates gene expression via recruitment of a histone deacetylase complex.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

Visual system homeobox 1 (Vsx1) is a paired-like:CVC homeodomain transcription factor that is expressed in a subset of retinal bipolar cells. Vsx1-null mice have previously been shown to have defects in bipolar cell terminal differentiation characterized by the reduced expression of four OFF bipolar cell-specific markers and electrophysiological defects in the OFF visual signaling pathway. The availability of recently identified bipolar cell markers enables a further characterization of the Vsx1-null mutant. I determined that Vsx1 is expressed in Type 7 ON bipolar cells and observed the upregulation of three cell markers: Cabp5, Chx10, and alpha-gustducin:GFP in this cell type in Vsx1-null mice. These data reveal a trend in which Vsx1 functions as a transcriptional repressor in Type 7 ON bipolar cells and as an activator in Type 2 OFF bipolar cells. Lastly, my data indicate that Vsx1 is required for the expression of two Type 3a bipolar cell markers, however, the mechanism by which it does so appears to be complex, as I was unable to detect Vsx1 protein or reporter gene expression in this cell type. / Graduate

Like many Gram-negative bacteria, the food-borne pathogen Yersinia pseudotuberculosis harbours different regulatory mechanisms to maintain an intact bacterial envelope especially during exposure to extracytoplasmic stress (ECS). The CpxA-CpxR two component regulatory system is one such ECS-responsive regulatory mechanism. Activation of CpxA-CpxR two-component regulatory system (TCRS) accumulates phosphorylated CpxR (CpxR~P), which not only up-regulates various factors that are designed to maintain envelope integrity, but also down-regulates key determinants of bacterial virulence. Y. pseudotuberculosis establishes close host cell contact in part through the expression of the invasin adhesin. Invasin expression is positively regulated by the transcriptional regulator RovA, which in turn is negatively regulated in response to nutrient stress by a second transcriptional regulator RovM. In Y. pseudotuberculosis, loss of CpxA phosphatase activity accumulates CpxR~P, and this represses both rovA and inv transcription directly, or indirectly via activation of rovM transcription. It is now of interest to understand the molecular mechanism behind how CpxR~P regulates gene transcription both positively and negatively. A type III secretion system (T3SS) is a highly conserved multi-protein secretion system used by many Gram-negative bacteria to secrete protein cargo that counteracts the effects of a host cell emitted anti-bacterial activity. A typical set of proteins that make-up a functional T3SS includes structural proteins, translocators, effectors and regulatory proteins. Accumulation of CpxR~P was shown to repress the plasmid encoded Ysc-Yop T3SS of Y. pseudotuberculosis. Although yet to be confirmed experimentally, promoter-CpxR~P binding studies indicate multiple modes of regulatory control that for example, could influence levels of the plasmid-encoded Ysc-Yop system transcriptional activator, LcrF, and the chromosomal encoded negative regulators YmoA and YtxR.  Regulatory processes of TCRS involve transient molecular interactions between different proteins and also protein with DNA. Protein-protein interaction studies using the BACTH assay showed that it can be useful in analysing the molecular interactions involving the N-terminal domain of CpxR, while the λcI homodimerization assay can be useful in analysing molecular interactions involving the C-terminal domain of CpxR. Therefore, in combination with other biochemical and physiological tests, these hybrid-based assays can be useful in dissecting molecular contacts that can be helpful in exploring the mechanism behind CpxR~P mediated transcriptional regulation. In conclusion, this work uncovered direct involvement of CpxR~P in down-regulating virulence in Yersinia pseudotuberculosis. It also utilised genetic mutation and explored different protein-protein interaction assays to begin to investigate the mechanism behind the positive and negative regulation of gene expression mediated through active CpxR~P.

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.