Meiosis requires cells to undergo two successive rounds of division without an intermediate synthesis or S-phase. This results in a gamete with half the number of chromosomes that can then combine with those of another gamete to begin the development of a new offspring. Once an oocyte enters meiosis it is arrested in late prophase I and remains in that state until the animal reaches sexual maturity and undergoes ovulation. Upon hormonal stimuli, the oocyte is released from its first meiotic arrest, undergoes oocyte maturation, arrests in metaphase II and is ovulated to await fertilisation. The process of oocyte maturation is governed by the interaction of many different protein kinases, phosphatases and proteases. How these proteins co-localise with each other during oocyte maturation is a central aim of this thesis. One central protein is maturation promoting factor or MPF. MPF is composed of a catalytic subunit, cdc2 and a regulatory subunit, cyclin B1. Throughout oocyte maturation MPF activity is dynamic and changes in MPF activity coordinate the progression of oocyte maturation. The ubiquitin-proteasome pathway (UPP) plays a role in regulating the activity of MPF at specific times during oocyte maturation by targeting and degrading the cyclin B1 subunit. In this thesis the 20S proteasome ‘core’ of the 26S proteasome was localised in bovine and murine oocytes undergoing maturation in vitro. The 20S proteasome was found in the germinal vesicle prior to maturation in both species. Once germinal vesicle breakdown occurred, the proteasome consistently localised around the developing spindle in both species. Cyclin B1, an important target of the UPP was also localised in murine oocytes to see how the UPP interacted with its substrates during oocyte maturation. Cyclin B1 was found to have similar localisation patterns throughout oocyte maturation to the 20S proteasome. This demonstrates that the 26S proteasome moves to the location of its substrates in order to degrade them. To confirm the role of the UPP in cyclin B1 degradation, we inhibited proteasome function using MG132, a known inhibitor of proteasome function. Polar body 1 (PB1) extrusion declined significantly (P<0.05) in a dose dependent manner, confirming that the UPP is essential for the continuation of meiosis. Our localisation data indicated that cyclin B1 interacted with the UPP during the GV stage and around GVBD. Inhibition of the proteasome, again using MG132, prevented oocytes from undergoing GVBD and arrested them in the GV stage. This demonstrated that the UPP was essential for oocyte maturation as well as indicating a potential switch in substrate during this transition. The deubiquitinylating enzyme, Fat Facets in Mouse (FAM also know as USP9x), an antagonist of the UPP, was also localised in murine oocytes undergoing maturation in vitro. FAM was found primarily in the cytoplasm prior to germinal vesicle breakdown however it relocalised to the developing spindle upon germinal vesicle breakdown. FAM is known to rescue a number of specific substrates from ubiquitinylation and degradation, one being I- catenin, a multifunctional protein, not previously implicated in oocyte maturation. Localisation of I-catenin during oocyte maturation revealed distinct staining at specific stages of meiosis. Staining was observed on the plasma membrane and in the GV prior to maturation. During MI and MII it appeared to co-localise with FAM. When homologous chromosomes divided at AI/TI I-catenin distinctly localised to the cleavage furrow indicating its involvement during cytokinesis. To determine if FAM activity was essential for oocyte maturation we inhibited FAM function by microinjecting anti-FAM serum into eggs post GVBD. Inhibition of FAM prevented PB1 extrusion. Some oocytes appeared to attempt PB1 but failed to complete (Hoechst staining of the chromatin revealed these cells remained in MI). This finding strongly indicates that FAM maybe rescuing I-catenin from degradation during MI until it is needed at AI/TI. However further examination of this interaction are needed to clarify the data. This research demonstrates that regulation of turnover of key proteins is essential for progression through oocyte maturation. It also demonstrates that multiple pathways may regulate the same processes. This may add to the complexity of studying the cell, but it also demonstrates the elegance of a highly regulated and sophisticated system.

Thesis (Ph. D.)--Oregon Graduate Center, 1989.

B lymphocytes produce a wide array of antibodies to recognize a countless number of antigens. This highly diverse repertoire is produced during B cell development in the bone marrow from the immunoglobulin heavy chain (Igh) and light chain (Igk and Igl) loci. The mouse Igh is a large (~3Mb) multigene locus that contains 195 variable (V), 10 diversity (D) and 4 joining (J) genes that undergo developmentally regulated V(D)J recombination to produce the variable region of the antibody. Gene expression depends on spatial organisation of chromatin. To ensure that all V genes have a chance to recombine, they are brought into physical proximity to the D-J region by locus contraction and DNA looping. Not all V genes recombine with equal frequencies and we aim to investigate how dynamic changes in 3D structure of the Igh locus facilitate V(D)J recombination. Chromosome conformation capture techniques have revolutionised studies of genome conformation. I have applied a novel form of enriched Hi-C to study both intra-locus (cis) and genome-wide (trans) interactions of the immunoglobulin loci in pro-B and pre-B cells. This method provides a higher resolution than Hi-C and is less biased than 4C and 5C. I have mapped all cis interactions within the Igh locus to produce a comprehensive view of the structure of the locus prior to recombination. This approach has shown that the 3’ superanchor (3’CBEs) and the Intergenic Control Region 1 (IGCR1) containing CTCF sites are the two most interacting regions in the locus making long-range contacts with all V genes. A second major conformational feature is that the distal V genes form a large tightly looped domain forming the centre of mass of the locus to which the 3’CBEs and IGCR1 loop. Thanks to a collaboration on polymer modelling, 5000 single conformations were simulated based on the ensemble Hi-C data. This showed that every structure is different, supporting a model of dynamic and flexible organisation of the locus rather than hierarchical subdomains therein. Moreover, there is only a slight trend for V genes interacting more often with the D-J region to have higher recombination scores, supporting an ‘equal opportunity for all’ model in which participation of V genes in V(D)J recombination is not constrained by linear genomic distance from the DJ region. Nevertheless, CTCF binding level does contribute to V gene recombination frequency. I have also discovered that Igh and Igk loci participate in a highly specialised network of genome-wide (trans) interactions involving genes encoding B cell-specific factors essential for activation and maintenance of B cell identity, including Pax5, Foxo1, Ebf1, and Runx1. I have validated these by 3D DNA FISH and found that at the pro-B cell stage the Igh is involved in many trans interactions, whereas Igk does not make any contacts. In contrast, Igk gains numerous trans interactions at the pre-B cell stage, many of which overlap with the interactions Igh participates in at both developmental stages. Together, these findings reveal a complex developmentally regulated orchestration of genome conformation changes that underpins B cell development.

<p dir="ltr">Angiosarcoma (AS) is a rare, understudied cancer that arises from endothelial cells with an extremely poor prognosis. More research is necessary to understand AS pathogenesis, which will lead to the development of novel therapies to improve patient survival. Evidence from our lab highlights the importance of miRNAs in disease progression by demonstrating that endothelial cell-specific loss of mature miRNAs drives AS in mice. Furthermore, individual miRNAs have been characterized as tumor suppressors in AS. Taken together, this underscores the role of miRNAs in AS pathogenesis and suggests that they are an underexplored therapeutic strategy that could be efficacious in treating this cancer. Due to the evidence that miRNA loss is a driver of AS, we hypothesized that global miRNA enhancement would reduce cancer phenotypes. We interrogated this question through the use of a small molecule enhancer of RNAi, enoxacin. We found that enoxacin robustly reduced cancer phenotypes, particularly in AS models driven by miRNA loss, and that enoxacin increased the expression of mature tumor-suppressing miRNAs. We then thoroughly characterized miR-497 in angiosarcoma, demonstrating that miR-497 overexpression ablated tumor formation in mice through the regulation of a network of target genes. Furthermore, we identified <i>Vat1</i> as a novel target gene of miR-497, and found that genetic and pharmacologic inhibition of <i>Vat1</i> reduced cell migration in AS. Overall, this work further highlights the important roles miRNAs play in AS pathogenesis, and points toward miRNAs as an exciting therapy that should be explored further.</p>

The immune system recognizes and eliminates an enormous array of pathogens due to the diverse antigen receptor repertoire of T and B lymphocytes. However, the development of lymphocytes bearing receptors with unique specificities requires the generation of programmed double strand breaks (DSB) coupled with bursts of proliferation, rendering lymphocytes susceptible to mutations and oncogenic transformation. Thus, mechanisms responsible for monitoring global genomic integrity, such as those coordinated by the ATM (ataxia-telangiectasia mutated) kinase, must be activated during lymphocyte development to limit the oncogenic potential of antigen receptor locus recombination. I show that ATM deficiency compromises TCRα recombination and the post-mitotic survival of T-cell receptor αβ (TCRαβ+) CD4+CD8+ (DP) thymocytes, providing a molecular and developmental basis for the immunodeficiency characteristic of ATM loss. Moreover, I show that in early thymocyte progenitors undergoing TCRβ recombination, ATM loss leads to cell cycle defects and developmental arrest, likely facilitating the acquisition of mutations that contribute to leukemogenesis. Using ATM deficiency as a murine model of T cell precursor acute lymphoblastic leukemia (T-ALL), I demonstrate that IL-7 signaling, a critical survival and proliferation signal during early stages of normal thymocyte development, is also required for leukemic maintenance. Moreover, we show for the first time that in normal and leukemic thymocyte precursors, interleukin 7 receptor (IL-7R) expression and function are controlled by Notch signaling, a key determinant of T cell fate. Collectively, these findings provide insight into the mechanisms by which ATM promotes normal lymphocyte development and protects from neoplastic transformation, while establishing the groundwork for assessing the molecular events that lead to the initiation and stepwise progression of T cell leukemogenesis.

Proteolytic processing of transmembrane receptors and ligands can have dramatic effects on cell signaling and subsequent cellular responses. Previous studies demonstrated that a disintegrin and metalloproteinase 10 (ADAM10) may cleave numerous B cell-expressed receptors, including the low affinity IgE receptor (CD23). However, lethality of ADAM10-deficient embryos has limited examination of these cleavage events in lymphocytes. To investigate their role in B cell development and function, we generated B cell-specific ADAM10 knockout mice. Intriguingly, deletion prevented development of the entire marginal zone B cell (MZB) lineage. Further analysis revealed that ADAM10 is required for S2 cleavage of the Notch2 receptor and initiation of Notch2 signaling, which is required for MZB development. Additionally, cleavage of CD23 was dramatically impaired in ADAM10-deficient B cells. This finding and results of ex vivo cleavage assays demonstrated that ADAM10 is the principal in vivo sheddase of CD23. Previous studies have demonstrated that Notch signaling and CD23 cleavage regulate antibody production. Accordingly, deletion of ADAM10 profoundly inhibited germinal center formation, and T-dependent and T-independent antibody responses to immunization, implicating ADAM10 as a novel regulator of adaptive immunity. Additionally, to determine the role of ADAM10 activity in hematopoiesis, we generated transgenic mice (A10Tg) that overexpress the protease on lymphoid and myeloid progenitors. Surprisingly, this markedly suppressed B2 cell development and promoted dramatic expansion of myeloid-derived suppressor cells (MDSCs) via a cell intrinsic mechanism. A10Tg MDSCs inhibited T cell proliferation and adoptive immunotherapy of B16 melanoma, resulting in exacerbated metastatic progression that was prevented by MDSC depletion. Thus, A10Tg mice represent a novel model for the examination of MDSC development and MDSC-mediated immune suppression in a tumor-free environment. Finally, hematopoietic stem cell cultures revealed that ADAM10 overexpression directs myeloid development by dysregulating Notch signaling via uncoupling the highly regulated proteolysis of Notch receptors. Collectively, these findings demonstrate that ADAM10 is a critical regulator of Notch signaling, B cell development, and MDSC expansion. Moreover, they have important implications for the treatment of numerous CD23 and Notch mediated pathologies, ranging from allergy to cancer.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

Early T-cell development is regulated by a complex interplay between transcription factors and developmental cues which ensure that functional T-cells are produced within the thymus. Early thymocytes integrate these signals in a step-wise fashion that progressively restricts their lineage potential as they transition through the early stages of T-cell development. Gene knockout studies have shown that the E-protein transcription factor HEB is required for normal thymocyte development. Furthermore, many additional key regulators such as Notch1 have been identified, but the connections among them and their specific roles in early T-cell development have not been well established. In this thesis, I set out to determine the specific roles of HEB at the beta-selection checkpoint and to establish connections between HEB and the key regulators within the gene regulatory network that orchestrates early T-cell development. To facilitate these studies, I generated a series of new mouse models including HEBAlt transgenic mice that express a short form of HEB called HEBAlt, which enabled me to answer specific questions and examine rare populations. First, my studies of HEB-/- mice allowed me to identify an early block in T-cell development, which was alleviated upon the addition of an HEBAlt transgene. Furthermore, I identified pTa and CD3e signalling as specific targets of HEBAlt during -selection. Second, my studies on HEB-/- mice revealed that they have a defect in T-cell commitment, with compromised Notch1 function and a tendency to become DN1-like cells. Moreover, the DN1-like cells could be induced to differentiate into thymic NK cells, revealing a role for HEB in the T/NK cell lineage decision. This study has revealed a new set of interactions among HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. Unexpectedly, my studies have also provided evidence for a role of HEBAlt in lymphomagenesis, highlighting the strict regulation of E-protein function that is necessary to ensure normal T-cell development.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.