No description available.

Enhancers are cis-regulatory sequences which promote the expression of target genes in a spatial and temporal fashion. They can be located within genes or between them and can act at distances of over 1 Mb. There are several different mechanisms by which enhancers regulate gene expression. Some, such as those regulating the Hox genes, are located close to each other in the genome in a structure referred to as a regulatory archipelago. These come together and act in combination to regulate gene expression, with different enhancer combinations resulting in different patterns of expression. On the other hand, enhancers can act individually, with designated enhancers responsible for regulating the expression of the same gene in different tissues or at different stages of development. Indeed, this is the case for the Sonic Hedgehog gene (Shh) where several different enhancers located within a gene sparse region referred to as a gene desert, act separately leading to Shh expression in areas such as the brain, the lungs, the notochord and neural tube and the limbs. Within the developing mouse embryo, Shh is expressed over roughly a two day period from E10 to E12 in a posterior distal region referred to as the Zone of Polarising Activity (ZPA). Ectopic expression in anterior regions has been observed in some common congenital diseases which affect the limbs, sometimes resulting in the formation of extra digits. The reason for this mis-expression is largely due to defects in the Shh limb enhancer commonly referred to as the Zone of Polarising Activity Regulatory Sequence (ZRS). Mutations within this highly conserved sequence create additional protein binding sites thus activating the enhancer in the wrong locations. The associated diseases are known collectively as the ZRS associated syndromes and can range from the less severe phenotype of preaxial polydactyly type II (characterised by an extra digit near the thumb) to the more severe Werner Mesomelic Syndrome (WMS), where patients present with a clear displacement of their tibia. The mechanism by which the ZRS functions is yet to be fully elucidated, with current studies producing conflicting data. What is known, is that the region encapsulating the Shh gene is highly compact, with both the gene and its enhancers located in a highly conserved Toplogical Associated Domain (TAD) as proven by Hi-C experiments. The boundaries of this domain are likely created by the binding of the protein CTCF to specified binding sites located at the either end of the locus. This restricts the ability of the enhancers to regulate the expression of genes outside the TAD. To study the exact mechanism by which the ZRS is activated and regulates Shh expression, the Hill laboratory has used cultured cell lines derived from the posterior regions of an E11.5 limb bud. Gene expression in these cells is highly reflective of the posterior limb bud, with the key exception being Shh, which is not expressed. However, using different drug treatments or biological manipulations Shh can be activated thereby making this the perfect system to analyse the mechanisms leading to Shh activation. In this investigation the cell lines were used to determine how the position of the ZRS changes upon activation. Using techniques such as Fluorescent in situ hybridisation (FISH) with either fosmid probes or directly labelled probes called MYtags, it was confirmed that the Shh locus is indeed highly compact in both Shh expressing and non-expressing cells. However, no differences were observed in terms of the distance between the ZRS and Shh between these two conditions in our cell lines. Next, both carbon copy chromosome conformation capture (5C) and circular chromosome conformation capture (4C) were used to look at changes to the Shh locus in different conditions. This confirmed Hi-C experiments and other recent publications suggesting that Shh is located within a TAD, the position of which is highly conserved between different conditions and cell lines. Furthermore, treatments activating the Shh gene resulted in significant deviations to the chromatin interactions within the locus suggesting a repositioning of structures when the gene is active. It is believed that the use of Shh inducible limb derived cell lines will prove extremely useful in future scientific endeavours to study the mechanisms of mammalian limb development. These provide a quick and easy means of accessing large numbers of Shh expressing cells, a feature which is increasingly important in an era where large cell numbers are needed for conducting chromosome conformation capture experiments such as Hi-C, 5C and 4C.

The Hedgehog (Hh) signaling pathway plays a critical role in modulating various developmental processes that requires fine tuning of the Hh signal, such that dysregulation can lead to cellular events involved in cancer. To elucidate the factors responsible for aberrant Hh activation and subsequent tumorigenesis, I investigated three distinct modulators of Hh signaling: (1) p53 tumour suppressor (2) primary cilia (3) PTHLH. During chondrocyte development, abnormal Hh signalling can result in benign cartilage tumours, called Enchondroma. As precursor lesions, enchondroma may progress to malignant neoplasia, collectively known as chondrosarcoma. Although the molecular events involved in this progression are poorly understood, inactivation of the p53 tumour suppressor has been identified in approximately one-third of chondrosarcoma. Using an enchondroma mouse model, I showed that p53 deficiency can cause chondrosarcoma to develop. The combined inhibitory effects of Hh and p53 pathways on the pro-apoptotic factor, IGFBP-3, suppressed apoptosis and was demonstrated to play a critical role in the progression to chondrosarcoma. The primary cilium is an organelle that serves as a signaling centre for the Hh pathway to allow for greater control of the signal output. Loss of primary cilia results in abnormal Hh signaling that is associated with cancer and various developmental defects. I observed a depletion of primary cilia in both human Chondrosarcoma and Enchondroma tumours compared to normal cartilage. Analysis of cilia-deficient mice revealed that defective ciliogenesis alone could lead to the formation of benign cartilage tumours. Furthermore, loss of primary cilia potentiated the effect of Hh signaling activation, revealing a novel role in cartilage tumorigenesis. Parathyroid-like hormone (PTHLH) is an essential inhibitor of the Hh pathway during chondrocyte development, however its function as a regulator of Hh in other tissue types are largely unknown. Through activation of PKA, PTHLH suppresses the activation of Gli transcription factors; downstream effectors of the Hh pathway. Using irradiated Ptch+/- mice that exhibit a high incidence of skin and brain tumours, I demonstrated that treatment with PTHLH agonist, PTH (1-34), results in inhibition of the Hh pathway, increased survival and a reduction in tumour incidence and size. Thus, PTH (1-34) may have therapeutic potential for Hhrelated cancers, especially given its known clinical safety in treating osteoporosis.

Metazoans use Hedgehog signaling to direct many stages of embryonic development, and deregulation of this pathway is implicated in many types of cancer. I investigated several steps of Hedgehog pathway transduction that were poorly understood in mechanistic terms. The mature Hedgehog ligand is produced by a self-proteolysis reaction that covalently attaches a cholesterol molecule to the signaling half of the protein. I showed that the catalytic cysteine forms a disulfide bridge that is essential for the folding and function of the C-terminal tail of Hedgehog, and identified two protein disulfide isomerases that remodel this bridge to free the catalytic thiol group after folding is complete. Using pulse chase assays to follow Hedgehog processing, I demonstrated that the self-proteolysis reaction takes place in the endoplasmic reticulum, that the cleaved C-terminal tail of Hedgehog is degraded before moving to the Golgi, and that Hedgehog mutants defective in processing get degraded in their entirety by the same route. Lipidated Hedgehog ligand requires the transmembrane protein Dispatched for secretion. I devised a system to test Dispatched function in cultured cells, and showed that some inactive Dispatched mutants fail to bind Hedgehog, while others bind more tightly than the wild type protein. Scube2 was implicated as a Hedgehog pathway component in zebrafish genetic studies. I showed that Scube2 is a secreted protein that binds Hedgehog via its cholesterol adduct and solubilizes it in aqueous media. Dispatched and Scube2 bind Hedgehog on opposing faces, and they function synergistically to release it from the membrane. Vertebrate Hedgehog signaling relies on intraflagellar transport through an antenna-like organelle called the primary cilium. The Hedgehog receptor Patched and transducer protein Smoothened localize to primary cilia in a mutually exclusive pattern, depending on Hedgehog ligand presence. I showed that cytoplasmic components of the pathway Suppressor of Fused (SuFu, a pathway inhibitor) and Glioma-associated oncogene transcription factors (the Gli family, the effectors of the pathway) localize to primary cilia and accumulate there when Smoothened is activated. SuFu and Gli form a complex that dissociates when the pathway is turned on, and this dissociation depends on trafficking through the cilium.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. Aberrant pathway activity can result in various developmental disorders and cancers. The Hh receptor Patched1 (Ptc1) is a negative regulator of the pathway and acts as a tumor suppressor. Our lab and others have shown that Suppressor of fused (Su(fu)) and Kinesin family member 7 (Kif7) are major negative regulators of the pathway that function downstream of Ptc1. Medulloblastoma (MB) is the most common malignant pediatric brain tumor originating from the cerebellum. Several forms of MB have been identified, with abnormal activation of the Hh pathway associated with one major subtype. These tumors commonly show inactivating mutations in PTCH1, whereas mutations in SU(FU) are more rare. Mouse models with deletion of Ptc1 or expression of a constitutively active form of Smoothened (Smo) exhibit elevated Hh pathway activity, leading to MB formation. In this study, I examined the complex roles of Su(fu) in the formation of MB alongside the inactivation of Ptc1, Kif7 and p53 in the cerebellum. Unlike Ptc1+/- mice, Su(fu)+/- mice do not develop MB, even upon exposure to DNA damaging agents (X-ray irradiation or chemical carcinogenesis), which dramatically increases MB incidence in Ptc1+/- mutants. However, Su(fu)+/-;p53-/- mice develop MB and these MB tumor cells exhibit loss of heterozygosity of Su(fu), suggesting a protective role of p53 in tumor suppression in Su(fu) deficiency. Kif7+/- mice are not prone to tumorigenesis, even with deletion of p53. To bypass the embryonic lethality of Su(fu)- and Kif7-null mice, I generated neural stem cell-specific knockout mice, GFAP-Cre;Su(fu)f/f and GFAP-Cre;Kif7f/f. GFAP-Cre;Su(fu)f/f mice exhibit a severely disorganized cerebellum, with drastic up-regulation of p53 expression, and they survive past 1 year of age and do not develop MB. In contrast, GFAP-Cre;Kif7f/f mice appear grossly normal with only subtle cerebellar defects. These observations indicate that neither Su(fu) nor Kif7 inactivation is sufficient to drive tumorigenesis in the cerebellum. To investigate whether Su(fu) and Kif7 possess any overlapping tumor suppressor functions, I generated GFAP-Cre;Kif7f/f;Su(fu)f/f mice. Indeed, simultaneous loss of these negative regulators resulted in MB formation, which is correlated with increased Hh pathway activity as well as a lower level of p53 expression. Furthermore, I discovered a novel positive role for Su(fu) in MB development, as Su(fu) activity is required for robust formation of MB in Ptc1-deficient mice. Together, these data illustrate that Su(fu) plays a dual role in the genesis of MB, and suggest that the rare human MBs with SU(FU) mutations could be caused by simultaneous deletion of TP53 or KIF7.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. Aberrant pathway activity can result in various developmental disorders and cancers. The Hh receptor Patched1 (Ptc1) is a negative regulator of the pathway and acts as a tumor suppressor. Our lab and others have shown that Suppressor of fused (Su(fu)) and Kinesin family member 7 (Kif7) are major negative regulators of the pathway that function downstream of Ptc1. Medulloblastoma (MB) is the most common malignant pediatric brain tumor originating from the cerebellum. Several forms of MB have been identified, with abnormal activation of the Hh pathway associated with one major subtype. These tumors commonly show inactivating mutations in PTCH1, whereas mutations in SU(FU) are more rare. Mouse models with deletion of Ptc1 or expression of a constitutively active form of Smoothened (Smo) exhibit elevated Hh pathway activity, leading to MB formation. In this study, I examined the complex roles of Su(fu) in the formation of MB alongside the inactivation of Ptc1, Kif7 and p53 in the cerebellum. Unlike Ptc1+/- mice, Su(fu)+/- mice do not develop MB, even upon exposure to DNA damaging agents (X-ray irradiation or chemical carcinogenesis), which dramatically increases MB incidence in Ptc1+/- mutants. However, Su(fu)+/-;p53-/- mice develop MB and these MB tumor cells exhibit loss of heterozygosity of Su(fu), suggesting a protective role of p53 in tumor suppression in Su(fu) deficiency. Kif7+/- mice are not prone to tumorigenesis, even with deletion of p53. To bypass the embryonic lethality of Su(fu)- and Kif7-null mice, I generated neural stem cell-specific knockout mice, GFAP-Cre;Su(fu)f/f and GFAP-Cre;Kif7f/f. GFAP-Cre;Su(fu)f/f mice exhibit a severely disorganized cerebellum, with drastic up-regulation of p53 expression, and they survive past 1 year of age and do not develop MB. In contrast, GFAP-Cre;Kif7f/f mice appear grossly normal with only subtle cerebellar defects. These observations indicate that neither Su(fu) nor Kif7 inactivation is sufficient to drive tumorigenesis in the cerebellum. To investigate whether Su(fu) and Kif7 possess any overlapping tumor suppressor functions, I generated GFAP-Cre;Kif7f/f;Su(fu)f/f mice. Indeed, simultaneous loss of these negative regulators resulted in MB formation, which is correlated with increased Hh pathway activity as well as a lower level of p53 expression. Furthermore, I discovered a novel positive role for Su(fu) in MB development, as Su(fu) activity is required for robust formation of MB in Ptc1-deficient mice. Together, these data illustrate that Su(fu) plays a dual role in the genesis of MB, and suggest that the rare human MBs with SU(FU) mutations could be caused by simultaneous deletion of TP53 or KIF7.

No description available.

The Hedgehog (Hh) signaling pathway plays a critical role in modulating various developmental processes that requires fine tuning of the Hh signal, such that dysregulation can lead to cellular events involved in cancer. To elucidate the factors responsible for aberrant Hh activation and subsequent tumorigenesis, I investigated three distinct modulators of Hh signaling: (1) p53 tumour suppressor (2) primary cilia (3) PTHLH. During chondrocyte development, abnormal Hh signalling can result in benign cartilage tumours, called Enchondroma. As precursor lesions, enchondroma may progress to malignant neoplasia, collectively known as chondrosarcoma. Although the molecular events involved in this progression are poorly understood, inactivation of the p53 tumour suppressor has been identified in approximately one-third of chondrosarcoma. Using an enchondroma mouse model, I showed that p53 deficiency can cause chondrosarcoma to develop. The combined inhibitory effects of Hh and p53 pathways on the pro-apoptotic factor, IGFBP-3, suppressed apoptosis and was demonstrated to play a critical role in the progression to chondrosarcoma. The primary cilium is an organelle that serves as a signaling centre for the Hh pathway to allow for greater control of the signal output. Loss of primary cilia results in abnormal Hh signaling that is associated with cancer and various developmental defects. I observed a depletion of primary cilia in both human Chondrosarcoma and Enchondroma tumours compared to normal cartilage. Analysis of cilia-deficient mice revealed that defective ciliogenesis alone could lead to the formation of benign cartilage tumours. Furthermore, loss of primary cilia potentiated the effect of Hh signaling activation, revealing a novel role in cartilage tumorigenesis. Parathyroid-like hormone (PTHLH) is an essential inhibitor of the Hh pathway during chondrocyte development, however its function as a regulator of Hh in other tissue types are largely unknown. Through activation of PKA, PTHLH suppresses the activation of Gli transcription factors; downstream effectors of the Hh pathway. Using irradiated Ptch+/- mice that exhibit a high incidence of skin and brain tumours, I demonstrated that treatment with PTHLH agonist, PTH (1-34), results in inhibition of the Hh pathway, increased survival and a reduction in tumour incidence and size. Thus, PTH (1-34) may have therapeutic potential for Hhrelated cancers, especially given its known clinical safety in treating osteoporosis.

Thèse (Ph. D.)--Université Laval, 2009. / Résumés en français et en anglais. Bibliogr.: f. 193-231. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Techn. University, Diss., 2004--Dresden.