Aberrant expression of Platelet-derived growth factor A (PDGF-A) and PDGF receptor-β (PDGFR-β) play critical roles in the angiogenesis and proliferation of several malignancies. In this dissertation I explore the transcriptional regulatory role of the Gquadruplex- forming regions in the promoters of human PDGF-A and PDGFR-β, and identify new targets for developing small molecules to modulate their expression in tumors. For PDGF-A promoter, our studies focus on two essential nuclease hypersensitive elements, NHE(PDGF-A) and 5´-end far upstream 5´-SHS. The structural aspects of the intramolecular G-quadruplexes formed in NHE(PDGF-A) and the ligands to stabilize these secondary DNA structures have been investigated by using singlestranded and duplex DNA of the NHE(PDGF-A). We demonstrate that the G-quadruplexinteractive compound, TMPyP4, can selectively inhibit the basal promoter activity of PDGF-A, suggesting that the NHE(PDGF-A) G-quadruplex acts as a repressor in PDGF-A transcription. We also found that the 5´-SHS G-rich strand oligomer can invade the NHE(PDGF-A) and form a unique three-stranded complex in supercoiled plasmids, which is facilitated by potassium ions and TMPyP4. Therefore, we propose a novel molecular mechanism for transcriptional silencing of the NHE(PDGF-A) by 5´-SHS in the PDGF-A promoter, in that the formation of G-quadruplex in the NHE(PDGF-A) provides a platform for the G-rich strand of 5´-SHS to invade and form a partial duplex DNA with the C-rich strand of the NHE(PDGF-A), resulting in displacement of hnRNP K and thus transcription silencing. Prior to the studies describe here, the promoter of human PDGFR-β had not been identified. Herein, we have cloned and characterized the first functional promoter of human PDGFR-β gene. A crucial highly GC-rich region (NHE(PDGFR-β)) in the human PDGFR-β promoter has been identified by its hypersensitivity to the S1 nuclease. Further studies demonstrate that stable G-quadruplex structures can form in the G-rich strand of NHE(PDGFR-β). The G-quadruplex-interactive molecule, telomestatin, can selectively stabilize G-quadruplexes formed in the human PDGFR-β promoter and inhibit its expression in Daoy cells. On the basis of these results, we propose that ligandmediated stabilization of the G-quadruplex structure in the proximal promoter region of human PDGF-A or PDGFR-β can be used to modulate the expression of these protooncogenes.

The confluence of next generation DNA sequencing and synthesis when combined with the application of concepts such as standardization and modular design has led to the genesis of a new discipline. The nascent field of Synthetic Biology concerns the rational design and construction of genetic circuits, pathways, machines and eventually whole organisms. The immaturity of this field dictates that early research efforts, including this Thesis, describe foundational work towards the creation of tools which make biology more amenable to being engineered. The first part of this Thesis describes an attempt to standardize the measurement of transcriptional promoter activity in E. coli. A method to measure in vivo promoter activity was developed for E. coli and tested in a multi-institution trial. Comparable results were achieved with less than a two-fold range for the measured promoters across eight laboratories. A standardized measurement kit was created and distributed for use by the teams participating in the 2008 international Genetically Engineered Machines competition. Techniques learned measuring the activity of E. coli promoters were applied to a collection of S. cerevisiae strains. Several promoters were measured in synthetic dextrose media and ADH1 was measured in multiple media conditions. The outcome of these experiments is to consider proposing ADH1as the reference promoter in S. cerevisiae. The second aspect of this Thesis describes the construction of artificial organelles in S. cerevisiae. Artificial organelles hold the prospect of being able to insulate synthetic genetic pathways from the cell. Two proteins are essential for the biogenesis of the peroxisome organelle in humans and yeast, Pex3p and Pex19p. Pex3p functions as a peroxisomal membrane receptor for Pex19p, while Pex19p shuttles other peroxisomal proteins to the membrane, including Pex3p, creating a feedback loop. Human Pex19p has previously been shown to dock to yeast Pex3p and a version of yeast Pex19p has been shown to work with human Pex3p as a high degree of evolutionary conservation exists between these proteins. Because of these inter-species protein docking characteristics, there exists the possibility of creating bimodality: the ambition of the work was therefore to create a cell strain which possessed both synthetic “humanized” and natural yeast peroxisomes. An S. cerevisiae BY4741a derivative strain was engineered with fluorophore tagged versions of human (CFP) and yeast (YFP) Pex3p and untagged yeast and human Pex19p proteins. The results indicated the creation of a single population of peroxisomes when a measure of fluorescently imaged CFP and YFP peroxisomes were plotted on a scatter plot. A log of the ratio of CFP to YFP peroxisomes was plotted on a histogram and a normal distribution was found to best fit the curve, indicating a lack of bimodality. Finally, microscopy images of this strain were reviewed and by visual inspection, showed no evidence of distinct human or yeast peroxisomes. This experiment therefore produced no evidence of bimodality when examining the interactions of human and yeast Pex3p and Pex19p proteins. However, the four proteins were shown to interact closely to produce a single population of chimeric human-yeast peroxisomes. The peroxisome-deficient mutant phonotype strain was rescued using human Pex3p and Pex19p.

No description available.

The regulation of the immune system is complex, with many factors involved in controlling immune cell development, activation and homeostasis. These factors include neuropeptides as well as classic immunoregulatory molecules such as cytokines, chemokines and hormones. Neuropeptides and tachykinins in particular are known to be involved in immune response modulation through a cascade of events including vasodilation, plasma extravasation, the activation of immune cells, the secretion of pro-inflammatory cytokines and the recruitment of more immune cells. Furthermore, there is growing evidence that tachykinins play a role in hematopoiesis with Substance P as the proposed effector molecule. In 2000, our lab discovered a new tachykinin with remarkable structural similarity to SP and SP-like neurokinin receptor binding affinity. This molecule was designated Hemokinin-1 due to its expression in hematopoietic cells and its function in B cell development. Further gene expression analysis of HK-1 reveals a wide expression pattern although HK-1 transcripts are found predominantly in peripheral tissues while SP is mainly expressed in neuronal tissue. Based on this differential expression pattern, it has been suggested that HK-1 may act as the peripheral tachykinin and may have functions distinct from SP. In addition, given the crossreactivity of the SP antibodies to HK-1, it is important to determine whether HK-1 is the actual mediator of some functions previously attributed to SP. In this thesis, we examine the differential expression pattern of HK-1 to determine molecular mechanisms of regulation of HK-1 transcription and ultimately provide clues to its function in the immune system. In our analysis of the HK-1 promoter, we found a major difference in the basic transcriptional control of HK-1 and SP at the level of transcription initiation and identified several transcription factors including CREB and NFκB involved in regulating TAC4 gene expression in immune cells. Data presented in this thesis also reveal that the HK-1 gene is a direct target of Early B-cell Factor, a transcription factor known to activate B cell-specific genes as well as genes involved in adipogenesis and neuronal development. Our results show EBF regulates HK-1 gene expression in differentiating B cells as well as a monocytic cell line. Our data indicate EBF may also be responsible for the high levels of HK-1 transcript in the olfactory epithelium, suggesting a bridge between the nervous system and the immune system.

HOX11 is a developmental regulator that plays a crucial role in the normal development of the spleen and is also aberrantly activated by the t(10;14)(q24;q11) and variant t(7;10)(q35;q24) translocations in a subset of T-cell acute lymphoblastic leukaemias (TALLs). The recent finding that HOX11 is deregulated in up to 40% of childhood TALLs when abnormalities not detected by cytogenetics are included, suggests that the over-expression of HOX11 and subsequent deregulation of downstream target genes are critical events in the progression of this tumour type. To date, three candidate HOX11 target genes have been reported, two of which are Aldehyde Dehydrogenase 1a1 (ALDH1A1) and Four and a Half LIM domain Protein 1 (FHL1). This investigation focused on two aspects of HOX11 function, namely its roles as a transcriptional regulator and as a nuclear oncoprotein capable of inducing neoplastic transformation. More specifically, we sought to further understand the role of HOX11 in tumorigenesis by 1) Confirming target gene status of ALDH1A1 and FHL1 by assessing whether their proximal promoter regions are transcriptionally regulated by HOX11, 2) Investigating the regulatory elements/transcriptional complexes involved in the response of ALDH1A1 to HOX11 in both a T-cell and an erythroid cell line in order to gain an insight into the mechanism(s) responsible for mediating a HOX11 activity and 3) Assessing the ability of ALDH1A1 and FHL1 to perturb normal patterns of haematopoiesis, on the basis that the transforming capabilities of HOX11 are thought to derive from its ability to affect haematopoietic cell differentiation. To confirm ALDH1A1 and FHL1 as target genes, they were both characterised in terms of the ability of their proximal promoters to be transcriptionally regulated by HOX11 using luciferase reporter assays. Significant repression of the proximal promoters of ALDH1A1 and FHL1 by HOX11 was observed in PER-117 T-cells which provided further evidence for their status as target genes. In the case of ALDH1A1, a CCAAT box (-74/-70bp) was identified as the primary cis-regulatory element involved in ALDH1A1 transcription and repression by HOX11 appeared to occur, either directly or indirectly, via interactions at the CCAAT box. Electromobility shift assays (EMSAs) revealed the disruption of a specific complex at this site by HOX11, which also altered the formation of complexes at a non-canonical TATA box (a GATA box at -34/-29bp). Significantly, HOX11 was shown to have the potential to interact with TFIIB, a member of the basal transcriptional complex. This, together with the presence of a TFIIB responsive element immediately 5’ of the GATA box, suggested that HOX11 may repress transcription by interfering with members of a preinitiation complex on the ALDH1A1 promoter. The transcriptional repression by HOX11 demonstrated in T-cells was dependent on DNA binding helix 3 of the homeodomain, suggesting that repression may require DNA binding. Alternatively, this region may be required for stable protein-protein interactions. In support of this, the in vitro association of HOX11 with TFIIB was disrupted upon deletion of helix 3, and the HOX11∆H3 mutant switched from a transcriptional repressor to a potent activator of transcription. Together, this data supports a model whereby HOX11 represses transcription by interfering with activation complexes at the CCAAT box and at the GATA box possibly via protein-protein interactions involving the homeodomain helix 3, whereas deletion of the region disables repressor-specific interactions, resulting in potent activation by HOX11. Luciferase reporter gene assays investigating the response of nested deletions of the ALDH1A1 promoter to HOX11 in the HEL900 erythroleukaemic cell line, also identified the CCAAT box (-74/-70bp) as the primary cis-regulatory element involved in ALDH1A1 transcription. However, in stark contrast to the its effect in T-cells, HOX11 was shown to activate transcription in the HEL cell line, both from the empty pGL3Basic luciferase reporter vector and from the ALDH1A1 promoter, in a manner independent of the homeodomain DNA binding helix 3. HOX11 thus appears to be a dichotomous regulator, capable of both transcriptional activation and repression depending on the circumstances. The mechanisms underlying these two functions are also appear to be distinct, with repression but not activation requiring the presence of homeodomain helix 3. ALDH1A1 encodes an enzyme involved in the irreversible conversion of retinaldehyde to the biologically active metabolite, retinoic acid (RA) and appears to be physiologically regulated by Hox11 in the developing spleen. Since RA is a potent modulator of cellular differentiation, proliferation and apoptosis, the dysregulation of RA synthesis is likely to have severe consequences for the cell and may constitute a mechanism whereby overexpression of HOX11 predisposes T-cells to malignant transformation. FHL1 also appears to have potential relevance to tumorigenesis, given that it encodes protein isoforms with suspected roles in transcriptional regulation. As a starting point to investigate a possible link between these HOX11 target genes and leukaemogenesis, the effect of overexpressing ALDH1A1 and FHL1 on murine haematopoiesis was assessed following reconstitution of lethally irradiated mice with retrovirally-transduced primary murine bone marrow cells. The enforced expression of ALDH1A1 in bone marrow was associated with a marked increase in myelopoiesis and a decrease in B and T-lymphopoiesis. By contrast, overexpression of FHL1 was not associated with perturbations in myelopoiesis or lymphopoiesis, although a slight increase in erythropoiesis was observed in the bone marrow. While further work is required to clarify the possible oncogenic roles of both of these HOX11 target genes, these findings have served to identify ALDH1A1 in particular, as a gene which could potentially be involved in HOX11-mediated tumorigenesis.

Les amplificateurs (aussi appelés par le terme anglais enhancers) ont été initialement identifiés comme des séquences d'ADN agissant en cis qui augmentent la transcription d'une manière qui est indépendante de leur orientation et de leur distance par rapport au site d’initiation de la transcription. En outre, les gènes d'identité cellulaire sont souvent associés à des regroupements ou clusters d’enhancers, structures également appelées super-enhancers, censés assurer une régulation correcte de l'expression des gènes tout au long du développement et de la différenciation des cellules. Pour mieux comprendre la régulation des gènes à partir de ces réseaux régulateurs complexes, nous avons étudié la régulation du gène Ikzf1 qui code pour un facteur de transcription essentiel à la différenciation lymphoïde et également impliqué dans la leucémogenèse. En combinant différents types de données épigénomiques, nous avons privilégié l’étude d’un élément enhancer situé à 120 kb en amont du gène Ikzf1. Nous avons trouvé que la délétion de l'enhancer IkE120 entraine une réduction significative de l'ARNm d’Ikzf1. Cependant, nous avons observé que la transcription immature ainsi que l’usage des promoteurs et exons alternatifs d’Ikzf1 sont différemment affectée dans les cellules délitées par IKE120. Ces résultats semblent indiquer que l'élément IkE120 pourrait avoir des fonctions supplémentaires au-delà de la seule régulation de l'initiation de la transcription. / Enhancers were originally identified as cis-acting DNA sequences that increase transcription in a manner that is independent of their orientation and distance relative to the RNA start site. In addition, cell identity genes are often associated with cluster of enhancers, also termed super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. To better understand gene regulation based on these complex regulatory networks, we studied the regulation of the Ikzf1 gene which encoded for a lymphoid-specific transcription factor essential for lymphoid differentiation and also involved in leukemogenesis. By combining different epigenomics data sets we prioritize an enhancer element located 120 kb upstream the IKZF1 gene. We found that deletion of the E120 enhancer resulted in significant reduction of Ikzf1 mRNA. However, we observed that immature transcription, promoter and exon usage were differentially affected in the IKE120-deleted cells. The results indicated that E120 element might have additional functions over solely regulating transcription initiation. We suggest that expression of some tissue-specific and cell identity genes might, at least partially, be regulated at the level of mRNA maturation and that components of enhancer’s clusters are directly involved in this process.

In transgenic research the precise control of transgene expression is crucial in order to obtain transformed organisms with expected desirable traits. A broad range of transgenic plants use the constitutive cauliflower mosaic virus (CaMV) 35S promoter to drive expression of selectable marker genes. Due to its strong enhancer function, this promoter can disturb the specificity of nearby eukaryotic promoters. When inserted immediately downstream of the 35S promoter in transformation vectors, special DNA sequences called insulators can prevent the influence of the CaMV35S promoter/enhancer on adjacent tissue-specific promoters for the transgene. Insulators occur naturally in organisms such as yeasts and animals but few insulators have been found in plants. Therefore, the goal of this study is to identify DNA sequences with insulator activity in Arabidopsis thaliana. A random oligonucleotide library was designed as an initial step to obtain potential insulators capable of blocking enhancer-promoter interactions in transgenic plants. Fragments from this library with insulator activity were identified and re-cloned into pB31, in order to confirm their activity. To date, one insulator sequence (CLO I-3) has been identified as likely possessing enhancer-blocking activity. Also, two other oligonucleotide sequences (CLO II-10 and CLO III-78) may possess insulator activity but more sampling is needed to confirm their activity. Further studies are needed to validate the function of plant insulator(s) and characterize their associated proteins.

Thesis (MSc. (Microbiology)) -- University of Limpopo, 2015 / S. cerevisiae has many properties which have made it the preferred host for the expression and production of a number of recombinant proteins. Xylose is the second most abundant sugar in nature and S. cerevisiae has been engineered to grow on this abundant sugar. Therefore, identifying S. cerevisiae promoters that are strongly induced during growth on xylose will be important in the production of recombinant proteins for the biofuel and other industries. Since xylose is not a native substrate for S. cerevisiae, it is not known how S. cerevisiae promoters will react during growth on xylose. The objective of the study was to evaluate the expression of a reporter gene, the Trichoderma reesei xylanase 2 (XYN2), under the control of five commonly used expression promoters (GPD3, ENO2, PGK1, ADH2 and YG100). Five episomal expression vectors were constructed for this purpose. These vectors were transformed to a recombinant xylose utilizing S. cerevisiae. Xylanase activity assays were used to determine the expression level from each of these promoters. The PGK1 promoter was observed to be the strongest promoter with average activity/OD of 130 nkat/ml/OD on both xylose and glucose. The GPD3 promoter showed the highest average activity/OD of 150 nkat/ml, but xylanase was only produced during growth on glucose. The data presented show that xylose is not a better carbon source than glucose for recombinant protein production in terms of the S. cerevisie promoters evaluated. Further research is required to obtain a yeast strain that grows well on xylose and promoters that show higher level on protein production. Keywords: xylose, promoter, expression, recombinant, S. cerevisiae

Previously we found that 3,4-(methylenedioxy)-1-(2′,3′- epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells. In this study, we further investigated which caspases were activated by safrole oxide during the apoptosis. The data showed that the activity of caspase-3, -8, and -9 was significantly enhanced by the compound, which suggested that safrole oxide might be used as a caspase promoter to initiate lung cancer cell apoptosis.

No description available.