Browsing by Subject "Molecular, Cellular, Developmental Biology and Genetics"

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The activin ligand dawdle links diet and metabolism via receptor isoform-specific signaling in Drosophila.
(2009-07) Jensen, Philip Anthony
Abstract not available.
Activin signaling promotes the competence of the prothoracic gland during Drosophila metamorphosis
(2010-08) Gibbens, Ying Ye
In insects, the timing of metamorphosis is modulated by a large titer of the steroid hormone ecdysone produced by the prothoracic gland. However, the molecular mechanisms that control production of the ecdysone titer are not completely understood. Here we show that blocking Activin signaling in Drosophila prothoracic gland causes developmental arrest prior to metamorphosis. This defect is due to the absence of the ecdysone titer, a likely consequence of reduced expression of the ecdysone biosynthetic enzymes. We further demonstrate that Activin signaling may regulate the competence of the prothoracic gland to respond to prothoracicotropic hormone and insulin, two hormonal signals that have been shown to trigger ecdysone synthesis. These findings suggest that Activin signaling is required for insect metamorphosis by providing competence that allows tissue- and stage-specific response to metamorphic stimuli.
B cell receptor signaling in receptor editing and leukemia.
(2009-07) Ramsey, Laura Bailey
There are many unanswered questions in the B cell field, but one of paramount importance is how B cells are tolerized to avoid autoimmunity. The majority of developing B cells are probably self-reactive, and many that make it through B cell development to the periphery show evidence of receptor editing. The question remains as to how the B cells signal to re-induce the editing machinery, whether it is a positive reactivation signal or the release of an inhibitory tonic signal. We propose that it is the release of a tonic signal through the B cell receptor that inhibits recombination when the BCR is present on the surface, but when the BCR binds self-antigen it is internalized, the inhibitory signal is abrogated, and the recombination machinery is reactivated. We tested this hypothesis using several transgenic models, including RAG2-GFP mice, HEL Ig mice, and anti-κ mice. We also used several mice deficient in BCR signaling to test this hypothesis, and found that reduced BCR signaling induces more receptor editing. When we tested mice with excessive BCR signaling or pharmacologically mimicked the BCR signal, we found a decrease in receptor editing. These experiments provide compelling evidence for the inhibitory tonic signaling hypothesis and against the activation signaling hypothesis. Another question unanswered in the B cell field is how B cells are transformed into leukemia and lymphoma. There have been whole genome analysis studies to show that genes involved in the BCR signaling pathway are involved in many B cell malignancies, including leukemia, lymphoma and myeloma. Our studies in mice provide evidence that in combination with constitutive STAT5 activation, a loss of genes involved in B cell development ( ebf1 and pax5 ) or pre-BCR signaling ( blnk, PKCβ, and btk ) results in leukemia. These are genes that have been shown to be deleted in B cell acute lymphoblastic leukemia (ALL). We also demonstrate an increase the phospho-STAT5 levels in adult BCR-ABL + ALL patients. Herein we have developed a new mouse model for B-ALL that may be useful in testing and perfecting treatment protocols used on ALL patients.
The BCL6 transcriptional corepressor (BCOR) is required for multiple aspects of murine embryonic development.
(2011-05) Hamline, Michelle Yvonne
The BCL6 transcriptional corepressor (BCOR) is mutated in the human multisystemic developmental disorder Oculofaciocardiodental syndrome (OFCD). The aim of this thesis is to understand the repressive function of BCOR in embryonic development. To accomplish this, BCOR was first tandem affinity purified from human embryonic kidney cells and found to interact with chromatin modifying proteins and several transcription factors, suggesting a molecular mechanism by which BCOR effects repression. In addition, conditional overexpression and null Bcor alleles were created in mice to elucidate the in vivo role of BCOR. The conditional overexpression allele revealed tight control of Bcor transcript levels in B cells and a requirement for proper control of Bcor expression for embryonic viability. The conditional null allele revealed that Bcor is required in neural crest cells for craniofacial development, in hindlimb precursors for proper limb formation, and in cardiovascular progenitors for cardiovascular development and function. These findings provide important insights into the function of BCOR in embryonic development and will facilitate the future diagnosis and treatment of developmental disorders such as OFCD.
Cancer gene discovery using somatic transposon mutagenesis in the mouse and systems for validation of identified candidate cancer genes and pathways.
(2012-07) Moriarity, Branden Scott
The conditional Sleeping Beauty (SB) transposon mutagenesis system has proven to be a successful method for cancer gene identification in solid tumors. Using tissue specific Cre recombinases (TSP-Cre) to selectively activate SB mediated mutagenesis in tissues of interest has yielded numerous novel candidate cancer genes in leukemia/lymphoma, colorectal, liver, and pancreatic cancers. We implemented the SB system for the identification of genes involved in osteosarcoma (OS). OS is the most common cancer of the bone and third most common cancer in children and adolescents. The 5-year survival rate of OS patients is 60-70%, though with metastatic disease it drops to less than 20%-30%. Identification of genes responsible for OS development and metastasis has been difficult due to its genomic instability and subsequent complexity. Consequently, there is a pressing need to identify the genes and pathways governing OS development and metastasis. To this end, we performed a forward genetic screen utilizing the conditional Sleeping Beauty (SB) transposase and mutagenic T2/Onc transposon system in Osx-Cre mice on a predisposing Trp53LSL-R270H/+ or wild type background to induce random somatic mutations that induce OS development and metastasis. One hundred and nineteen OSs were isolated from 96 predisposed and 20 wild type mice undergoing SB mutagenesis, with a subset of animals harboring metastases to the liver and/or lungs. Over 100 candidate cancer genes, both known and novel, were identified by analysis of recurrent T2/Onc insertion sites. In order to validate the candidate cancer genes identified in our screen, we developed a recombinase-based system for efficient assembly of vectors to over express or knock down 1-6 genes in mammalian cells. Further, these vectors were constructed in Piggybac transposons to allow for stable and reversible integration of the vector via transposition. In addition to cDNA over expression or shRNA knock down, we also developed methods for generation and selection of candidate cancer gene knock out cell lines using TAL effector nucleases (TALENs).
Characterization of the Sleeping Beauty transposon system for gene therapy applications /
(2008-05) Score, Paul Rodrick
Science and medicine are merging in development of gene therapy techniques that will likely become a recognized method for the treatment of human disease within our lifetime. The basic goal of gene therapy is to deliver therapeutic genes to target cell populations, allowing the cells themselves to generate a therapeutic gene product, for example to provide a gene product which is missing in the patient. Most of the current gene therapy trials have been based on the use of viral vectors to deliver therapeutic genes. These viral vectors are being used because they have already evolved mechanisms to efficiently enter cells. Because of the complications inherent in the use of viruses for delivery, a nonviral approach would have several advantages if it could achieve similar levels of efficiency. To this end, I set forth to characterize the Sleeping Beauty (SB) transposon system as a potential tool for effective nonviral gene transfer and its eventual use in clinical gene therapy protocols. The SB transposon system consists of a DNA cargo, usually plasmid based, which in the presence of the transposase integrates into chromosomal DNA. One of the key concerns for any gene delivery system is its ability to function in cells that are not dividing, as many cells in the body that are potential targets for gene therapy are non-dividing. In a series of in vitro experiments utilizing various techniques to halt cell division, I determined that cell division is likely not necessary for SB-mediated integration and expression to occur. Secondly, when tracking expression in vivo, it is not possible to distinguish the amount of gene product produced from integrated vs. nonintegrated transposons. Differentiating between these two sources of transgene expression soon after delivery, will allow insight into transposition efficiency in vivo that can relate to its clinical use. Using LoxP recombination sites, a Cre recombinase inducible mouse strain and transposons carrying a murine erythropoietin gene (Epo), I was able to silence expression from nonintegrated transposons and quantify in vivo gene expression specifically from transposed sequences. Over-expression of erythropoietin in the murine model became an unexpected problem due to subsequent erythrocytosis. Delivery of plasmid DNA to the livers of mice results in an initial spike of transgene expression and when coupled with the subsequent ubiquitous expression of the Epo transgene, circulating Epo levels remained greatly elevated, leading to serious health complications and death. To circumvent the initial spike of EPO expression, an inducible promoter was constructed that responds to hypoxic conditions. In this way, expression of erythropoietin should be regulated to prevent over-expression. Insights gained from these studies will contribute to our understanding of the capabilities of the SB system and its potential application to the treatment of human disease in the future.
Chromosome cohesion and condensation in Saccharomyces cerevisiae.
(2011-08) Hsu, Wei-Shan
Sister chromatid cohesion and chromosome condensation are two essential cell cycle processes for maintaining genome stability. Pds1 is the only known regulator for maintaining cohesion between sister chromatids during S phase till anaphase onset by inhibiting Esp1 activity, which is important for preventing aberrant chromosome segregation. However, pds1 null yeast are viable and able to keep cohesion during S phase. This indicates a redundant pathway maintains sister chromatid cohesion in the absence of Pds1. We have identified the Pds1-independent mechanism involved in S phase sister chromatid cohesion. This mechanism requires the function of two B-type cyclins, Clb5 and Clb6, as well as Cdc28. When DNA replication is efficient, either the Pds1- or Clb5/Clb6-dependent mechanism is sufficient to maintain sister chromatid cohesion. However, both mechanisms are required for S phase cohesion under conditions of replication stress. Further investigation revealed that cells lacking Clb5 and Clb6 have reduced levels of chromatin associated cohesin under conditions of replication stress. Further, this cohesion defect requires spindle tension to be observed by examining TRP1 locus separation. In conclusion, yeast cells maintain sister chromatid cohesion by a Clb5/Clb6 dependent mechanism for efficiently loading cohesin onto chromatin during S phase as well as a Pds1 dependent mechanism for inhibiting the protease activity of Esp1 before anaphase onset. Chromosome condensation requires the function of the condesin complex. Recent studies mainly focused on the ATPase activity of condensin in introducing DNA supercoiling. We studied how condensin is regulated through the cell cycle. We found that the protein levels of condensin subunits are cell cycle regulated. Chromosomes condense in response to higher condensin protein abundance. Chromosome decondensation at the end of the cell cycle requires Smc4 proteolysis mediated by APC/C ubiquitin ligase. This Smc4 protein turnover needs the function of Mad2 in the absence of nocodazole, but Mad2 is dispensable for Smc4 proteolysis under conditions of pre-anaphase arrest in the presence of nocodazole. In addition to protein abundance, phospho modification of Smc4 by Cdc28 also regulates the timing of chromosome condensation. This phospho modification of Smc4 destabilized Smc4 protein as well as preventing premature chromosome condensation early in the cell cycle. Collectively, we studied the underlying mechanisms of two essential events for genome stability: sister chromatid cohesion and chromosome condensation. Both events are under regulation by multiple mechanisms to ensure the faithfulness. Understanding these mechanisms using budding yeast would avail against the human diseases caused by genome instability.
Cloning and characterization of MRI, a modulator of retroviral infection.
(2009-12) Agarwal, Sumit
Although significant progress has been made in elucidating the life cycle of HIV and identifying host cell proteins which interact with HIV, a much greater understanding of these processes is necessary in order to eradicate this disease. The studies presented in this dissertation further examine these processes and identifies a new host cell protein, MRI, which appears to be involved in the poorly understood step of viral uncoating.The first part of my project involved the creation, characterization, and utilization of a novel toxic retroviral vector encoding barnase, a non-specific protease derived from Bacillus amyloliquefaciens. In conjunction with an inhibitor of barnase (barstar), I showed that barnase-based retroviral vectors were capable of inducing cell death in a wide variety of cell types and that the presence of barstar was essential for the creation of high titer retroviral vectors. The second part of my project was to was to create a heavily mutagenized hamster cell line, V79-4, and repeatedly challenge this cell line with the toxic retroviral vector in order to isolate clonal populations of cells resistant to retrovirally mediated transduction. Ultimately, I isolated two cell lines, 31-2 and 67-1, which we characterized further. 31-2 was determined to be least five fold resistant to both MLV- and lenti-viral at a stage in viral replication post reverse transcription. 67-1 was minimally ten fold resistant to both MLV- and lenti-viral infection at a stage between viral entry and uncoating. We demonstrated that the mutation in 67-1 implicated the proteasome and identified a gene we designated MRI that reversed the inhibition of infection in the mutated 67-1 line. The implication of MRI in the retroviral lifecycle identifies a new protein that could facilitate the creation of novel host cell based therapy for HIV.
Compounds derived from birch trees that inhibit HIV-1 replication
(2011-02) Dorr, Casey R.
Human Immunodeficiency Virus type-1 (HIV-1) replication is introduced in Chapter 1 with an emphasis on the late phase of viral replication. Literature about the production of infectious HIV-1 is reviewed in Chapter 1 while focusing on targets for suppressive HIV-1 therapy. Chapter 2 describes the discovery of triterpene compounds derived from birch trees that inhibit HIV-1 replication reformatted from Bioorganic and Medicinal Chemistry Letters. Chapter 3 investigates the anti-HIV-1 mechanisms of the triterpenes described in Chapter 2. Virus release assays conclude that the triterpene compounds target and prevents cleavage of the HIV-1 Gag product CA-SP1. Virus release assays and transmission electron microscopy indicate that the triterpene compounds SY33 and Bevirimat have a secondary mechanism of action by causing 55 kDa Gag to accumulate in cells. The Gag accumulation in cells was observed in both wild-type HIV-1 and the mutant SP1-A1V. The SP1-A1V mutant causes a decrease in susceptibility, by viral replication and CA-SP1 processing, to SY33 and Bevirimat. An Epilogue describes recommended future experiments. Appendix A describes genotypic results of an experiment to select for HIV-1 resistant to SY33 and Bevirimat using wild type and SP1-A1V as the founder viruses. The data in Appendix A suggest SP1-A1V causes reduced susceptibility to SY33 and Bevirimat. Appendix B describes the initial discovery of fatty acid derivatives with anti-HIV-1 activity.
Dia2 mediates Mrc1 degradation to promote checkpoint recovery from DNA damage in Saccharomyces cerevisiae.
(2012-08) Fong, Chi Meng
Cell cycle progression is monitored by checkpoint pathways that pause the cell cycle when stress arises to threaten the integrity of the genome. Although activation of checkpoint pathways has been extensively studied, our understanding of how cells resume the cell cycle when the stress is resolved is relatively limited. In this thesis, I identify the F-box protein Dia2 as a novel player in the S-phase checkpoint recovery pathway. Dia2 is required for robust deactivation of the Rad53 checkpoint kinase and timely completion of DNA replication during recovery from DNA damage induced by methyl-methanesulfonate (MMS). Aiming to identify the substrate of SCFDia2 (Skp1/Cul1/F-box Dia2) in checkpoint recovery, I performed a genetic screen to identify suppressors of dia2-null cells. The screen identified a new checkpoint-defective allele of MRC1 truncated at the C-terminus. I found that checkpoint-defective mrc1 alleles suppress the MMS sensitivity and the checkpoint recovery defect of dia2-null cells. In addition, Dia2 is required for Mrc1 degradation during S-phase checkpoint recovery. Furthermore, induced degradation of checkpoint-functional Mrc1 is critical to checkpoint recovery of dia2-null cells. These data support a model in which Dia2 mediates Mrc1 degradation for cells to resume the cell cycle during recovery from MMS-induced DNA damage in S-phase.
The extreme C-terminus of human Topoisomerase IΙ alpha defines a novel bi-modular DNA tether essential for the formation of mitotic chromosomes.
(2012-05) Lane, Andrew Besançon
Topoisomerase II is the target of an important class of anti-cancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. We have determined the mechanism of Topo IIA recruitment to chromatin and provide new insight into the formation of mitotic chromosomes. We describe the first example of what is likely to be a widespread mechanism for recruitment of chromosomal proteins involving a bi-modular element consisting of an NLS and an associated DNA tether. Catalytically dead Topo IIA is successfully targeted to chromatin, but both the catalytic activity and the bi-modular targeting element are essential for mitotic chromosome formation. Because reduced strand passage activity protects cells from Topo IIA-targeted drugs, it is likely that mutations in the bi-modular element would lead to drug-resistance.
Functional characterization of the three isoforms of Fbw7 (F-box and WD repeat domain containing 7) in ubiquitin dependent proteolysis.
(2010-01) Zhang, Wei
Fbw7 is the F-box protein of SCFFbw7 E3 ubiquitin ligase, which specifically associates with the substrates to be ubiquitinated. Substrates of Fbw7 play important roles in cell cycle regulation, proliferation, signal transduction and metabolism, which are related to tumor formation, suggesting that Fbw7 functions as a tumor suppressor. Fbw7 has three splicing variants α, β and γ, and the biological function of each isoform is not well understood. Our lab is interested in how the Fbw7 isoforms regulate cyclin E proteolysis and the cell cycle. By using mammalian and insect cell culture systems, I demonstrate that the three isoforms can form homo- and heterodimers in vivo and in vitro. The dimerization domain is located immediately upstream of the F-box motif, and it is highly conserved in all Fbw7 homologues and other related F-box proteins, indicating the dimerization may be common feature of a subset of F-box proteins. Abolishment of dimerization inhibits cyclin E proteolysis and leads to a prolonged half-life of cyclin E, although it does not affect Fbw7 binding to cyclin E or to the Cul-Rbx1-Skp1 E3 catalytic module. Cyclin E accumulation can be commonly found in many primary tumors and cancer cell lines. These results suggest a novel mechanism of how F-box proteins recognize their substrates. Fbw7 isoforms show different protein stabilities, where the α isoform is stable, but the β and γ isoforms are not. The stability of the β and γ isoforms is largely controlled by their N- terminal unique region. In order to better understand the mechanism regulating their stability, we performed a yeast two hybrid screen and identified SLP1 (stomatin like protein 1) as an Fbw7γ isoform specific interacting protein. SLP1 binds to the unique region of γ isoform, and stabilizes γ. We find that Cdk2 promotes the degradation of both SLP1 and the γ isoform, and this function of Cdk2 is dependent on its kinase activity. SLP1 also physically interacts with Cdk2 through its membrane association domain. These results support a model in which Fbw7γ and SLP1 are coordinately targeted for ubiquitin mediated degradation by Cdk2.
Functional roles of the gap junction protein, Connexin43.4, during vertebrate development.
(2009-05) Hatler, Julia M.
During early embryonic development, compartments of cells must send and receive signals in order to coordinate activities such as proliferation, migration and differentiation. Cells regulate these activities inpart through cell-to-cell communication by sharing small signaling molecules and ions via Connexin (Cx)-based gap junction (GJ) channels that connect the cytoplasms of adjacent cells. Mouse knockout models of Cx proteins have demonstrated the necessity for GJ communication during development; cx45 knockouts are embryonic lethal and cx43 null embryos exhibit defects in heart development. However, the mechanisms by which GJ communication regulate early embryonic development, particularly in terms of axis specification and patterning, remain largely unexamined. Here, the role of zebrafish Cx43.4 (the ortholog of mammalian Cx45) in patterning the asymmetric left-right (L-R) axis of zebrafish was examined. L-R patterning is directed by ciliary beating that generates a leftward fluid flow in the mammalian node or in Kupffer's vesicle (KV), the related structure in zebrafish. Leftward nodal flow is required for normal asymmetric organ placement and function. Cx43.4 expression was detected in KV and morpholino (MO) knockdown of Cx43.4 resulted in randomized organ distribution and reversed asymmetric gene expression. The major finding of these knockdown experiments is that Cx43.4 is required for the morphogenesis of KV. Additionally, Cx43.4 hemichannels, rather than GJ communication, are sufficient to rescue the defects in asymmetric patterning. Finally, the function of Cx43.4 hemichannels, together with purinergic receptors, is required for the morphogenesis of the KV epithelium. These novel findings have important implications for both the communication and L-R development fields, as well as suggesting new roles for Cx proteins in the early development of a variety of epithelial tissues.
Gene therapy for Athabascan SCID
(2010-09) Multhaup, Megan Marie
Artemis is an endonuclease characterized as a key factor involved in both nonhomologous end joining (NHEJ) and variable (diversity) joining (V(D)J) recombination. Mutations in the gene encoding Artemis result in a radiationsensitive form of severe combined immunodeficiency (SCID) found at a high incidence in Athabascan-speaking Native Americans (SCID-A) and characterized by the absence of mature B and T lymphocytes. Early treatment is critical since otherwise the disease results in severe infections that ultimately lead to fatality at a young age. The current therapy for SCID-A is allogeneic hematopoeitic cell transplantation (HCT); however, HCT often results in incomplete reconstitution of B lymphocytes and may lead to complications such as graft versus host disease. Transplantation with genetically corrected autologous cells is an alternative approach that may provide improved treatment of SCID-A. Lentiviral vectors pseudotyped with VSV-G are compelling candidate vectors for gene transfer considering their high transduction efficiency and capability to mediate gene transfer in non-dividing cells populations, such as quiescent hematopoietic stem cells. Accordingly, I developed several lentiviral vectors for the transduction of human Artemis cDNA into hematopoeitic cells for the correction of a murine model of SCID-A. Upon characterization of these vectors I found that Artemis over-expression results in a decrease in cell survival due to genomic DNA fragmentation, cell cycle arrest, and ultimately apoptosis. These data emphasize the importance of transgene regulation and demonstrate the necessity of establishing conditions that provide Artemis expression at a level iv that is non-toxic yet sufficient to complement Artemis deficiency. To this end, I subsequently recovered and characterized the endogenous human Artemis promoter (APro) as a one-kilobase region located directly upstream of the human Artemis translational start site. APro conferred a moderate level of reporter gene expression in vitro and in vivo, including secondary mouse transplant recipients, thus demonstrating reliable expression after lentiviral gene transfer into hematopoeitic stem cells. Subsequently, I compared innate regulation of the human Artemis cDNA using its own endogenous promoter sequence to that of the strong EF1α and more moderate PGK promoter for the capacity to mediate correction of a murine model of Artemis deficiency presenting a B- T- phenotype and exhibiting no leakiness (mArt -/-). Transplantation with both APro-hArtemis and PGK-hArtemis transduced mArt -/- marrow led to complete reconstitution of the immune compartment in the recipient animals. Beginning at 8 weeks posttransplant, the recipient animals had wild-type levels of CD3+CD4+ and CD3+CD8+ T lymphocytes and B220+NK1.1- B lymphocytes, cell populations that are absent in mArt -/- immunodeficient mice. However, transplantation with EF1α-hArtemis transduced marrow did not support immune reconstitution, suggestive of cytotoxic effects caused by Artemis over-expression. AProhArtemis treated mice exhibited restored IgM and IgG responses against 4- hydroxyl-3-nitrophenylacetyl hapten conjugated-keyhole limpet hemocyanin as well as restored cellular immune function, as assessed by in vitro stimulation of isolated splenocytes with anti-CD3 or concanavalin A. These results demonstrate that the naturally regulated Artemis lentiviral vector effectively complemented murine SCID-A, contributing to the development and advancement of gene transfer as a clinically relevant and feasible approach for treatment of SCID-A in humans.
Histone methylation in polycomb gene silencing.
(2010-08) Joshi, Preeti Madhav
Polycomb (PcG) group proteins are chromatin-modifying factors that collaborate to repress gene expression during development. PcG proteins are crucial for silencing during animal embryogenesis, maintenance of stem cell populations and are also implicated in cancer epigenetics. PcG proteins operate in at least three molecular complexes: PHORC, PRC2 and PRC1. Current models suggest that PRC1 is recruited at target loci by PRC2 and is most directly responsible for gene silencing. Our study uses the Drosophila model system to investigate the precise role of PRC2 in PRC1 targeting. One key function of PRC2 is to catalyze methylation of histone H3 on lysine-27. A common model suggests that this creates a binding site for the recruitment of PRC1. The loss of PRC2 leads to disruption in PcG mediated silencing. We wished to assess if PRC1 is recruited at PcG targets by a direct interaction with PRC2 or by binding to the methyl mark. We have used enzyme-dead versions of PRC2 that can stably accumulate at target loci and shown that PRC1 is also found at these targets suggesting that the initial recruitment of PRC1 is independent of PRC2 catalytic function. We have also engineered a heterologous enzyme, called vSET, to create methylated histones in the absence of PRC2. This heterologous enzyme can be used to test for PRC1 accumulation without PRC2.
Identification and characterization of novel positive regulators of the Target of Rapamycin (TOR) pathway in Drosophila melanogaster
(2008-12) Goraksha-Hicks, Pankuri
Target of Rapamycin (TOR) is an evolutionary conserved pathway in eukaryotes for growth and proliferation. TOR pathway plays an important role for growth homeostasis by coupling nutritional and growth factor availability and cellular stress levels to both temporal and spatial aspects of growth. Aberrant TOR signaling results in dysregulation of growth leading to a number of human pathologies such as Tuberous Sclerosis, Peutz-Jegher's syndrome, cancers and neurological diseases such as Alzeihmer's. Inspite of the importance of TOR-mediated growth, there are a number of unanswered questions in the pathway such as unknown regulators and effectors of TOR and how exactly amino acids are sensed by TOR pathway. In order to understand the complexity of the TOR pathway and gain answers to some of the questions in the pathway, we undertook forward and reverse genetic approaches to identify novel positive regulators in the TOR pathway using Drosophila as a model. Our studies identify Rag GTPases to be important for amino acid sensing and upregulating TOR activity. We also present a possible mechanistic model for how Rag-GTPase mediated amino acid sensing may activate TOR pathway. The positive regulators identified in this study have aided our understanding of the important process of amino acid sensing at a cellular level and have contributed to furthering our knowledge of the TOR pathway.
Identification of microenvironment factors that regulate hematopoietic stem and progenitor cells.
(2009-01) Buckley, Shannon Mychel
The hematopoietic stem cell (HSC) maintains hematopoiesis throughout life. HSC are used to treat a number of hematopoietic disorders including leukemia and other hematopoietic malignancies, as well as immunodeficiencies. The first successful bone marrow (BM) transplantation was performed over 30 years ago. Even so, we still have an incomplete understanding of what regulates different aspects of HSC behavior. To repopulate the hematopoietic system, physicians initially used bone marrow (BM) as a cell source, and more recently granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood (PB) cells, and umbilical cord blood (UCB). Although HSC transplantation can cure a significant proportion of patients, limitations still remain. Histocompatibility complex leukocyte antigen (HLA) matching is imperative to reduce the risk of graft versus host disease, such that a suitable donor cannot be found for many patients. Near complete matching is required when adult sources of cells are used, whereas, some degree of HLA-mismatch is allowed when UCB grafts are used, even though a low incidence of severe graft versus host disease (GVHD) persists following UCB transplantation. One of the problems related to the use of UCB as cell source is the limited number of HSC per graft, such that one UCB unit is insufficient to treat adult patients. Therefore, many investigators have examined whether HSC can be expanded ex vivo, as this would circumvent the problem associated with the limited number of HSC present in UCB. However, robust methods for ex vivo maintenance, let alone HSC expansion, have not been developed. Understanding the mechanisms that regulate proliferation, self-renewal, and differentiation of HSC may yield means of HSC expansion in vitro, as well as, allow for a single cord blood graft to be used for an adult recipient, and aid in attempts for genetic modification of autologous HSC for therapy of genetic hematopoietic disorders. Theoretically, the potential to expand HSC in vitro could provide a limitless supply of HSC for therapy. In vivo, HSC receive signals from the local microenvironment in which they reside that regulate self-renewal vs. differentiation. In adults, HSC reside in the BM, in putative stem cell niches. The hypothesis of the HSC niche was first proposed in 1978 by Schofield, who proposed that cell contact is required between HSC and cells in the niche for HSC maintenance. Over the last 5 years, two different locations have been identified within the adult BM that may serve as the BM niche: the endosteal niche and the vascular niche. Even less is known regarding putative stem cell niches during development. During development hematopoiesis is initiated in the yolk sac and the aorta-gonad-mesenephros (AGM) region. Around embryonic day (E) 12 in mouse, HSC migrate to the fetal liver (FL) where extensive expansion of the HSC pool occurs and HSC differentiate to the lineage committed progenitors. HSC migrate from the liver to the BM just before birth. As each of these microenvironments plays a unique role in development of hematopoiesis, it is hypothesized that they may possess unique signals that govern different aspects of HSC development, self-renewal and differentiation. However, the nature of these signals is still poorly understood. The aim of this thesis was to identify candidate factors produced by the HSC niche that regulate HSC cell maintenance (and expansion). In Chapter 4 of this thesis I hypothesized that at least some factors produced in HSC niches responsible for HSC self-renewal would be soluble. A number of stromal cell lines have been generated from the AGM region, fetal liver and BM that support HSC in vitro. I used three stromal cell lines that preserve human and / or mouse HSC cultured in direct contact with the feeders, namely UG26-1B6, derived from the AGM region of E10.5 embryos; EL08-1D2, derived from fetal liver of E10.5 embryos; and AFT024, derived from fetal liver of E14.5 embryos. To determine if soluble factors secreted by these feeders could support HSC, I cultured HSC-enriched murine BM cells either in direct contact with or separated by a transwell from the feeder for 3 weeks, and tested the ability of the ex vivo maintained cells to competitively repopulate the hematopoietic system of lethally irradiated mice. I demonstrated that only UG26-1B6 cells support competitive repopulating HSC cultured separated from the feeder, and that UG26-1B6 cells must secrete one or more HSC-supportive factors that are not secreted by the EL08-1D2 feeder. In Chapter 5, I describe studies in which I compared the transcriptome of UG26-1B6 and EL08-1D2 cells, to identify candidate factors secreted by UG26-1B6 but not EL08-1D2 that support HSC maintenance/expansion in vitro. Eighteen candidate-secreted factors were identified. I describe initial studies to test if 3 of these 18 factors, Galectin-3, Tfpi, and SerpinE2, can support HSC ex vivo. In Chapter 6, I evaluated the effect of a fourth factor expressed significantly higher in UG26-1B6 than EL08-1D2 cells, Wnt5a, on HSC support in ex vivo cultures. I describe studies to determine if (1) addition of Wnt5a to the EL08-1D2-based cultures allows maintenance of HSC placed in transwells above the feeders and if addition of anti-Wnt5a antibodies to the UG26-1B6-based cultures prevented HSC persistence; (2) if Wnt5a can also be used in the absence of any stromal support to maintain HSC; (3) and to evaluate the mechanisms via which Wnt5a affects HSC. In Chapter 7 of the thesis, I evaluated the effect of bone morphogenic protein (BMP) antagonists on HSC maintenance / expansion. These studies were not strictly derived from the transcriptome studies described in Chapter 5, but followed from other studies in the lab demonstrating that BMPs play a key role in mesoderm specification and initiation of hematopoiesis during zebrafish development. I describe studies (1) wherein the effect of loss of two BMP antagonists, Twisted gastrulation (Twsg1) and Chordin (Chrd), on adult hematopoiesis is evaluated, (2) and studies wherein the effect of addition of Twsg1 and Chrd to ex vivo feeder-free cultures on the repopulation ability of HSC is being tested.
Identification of novel genes involved in prostate and Schwann cell malignancies utilizing a sleeping beauty transposon somatic cell mutagenesis screen
(2011-06) Rahrmann, Eric Patrick
The focus of this thesis was identification of new genes and genetic pathways altered in prostate cancer and malignant peripheral nerve sheath tumor formation utilizing a Sleeping Beauty somatic mutagenesis forward genetic screen. Mutagenesis of prostate epithelial cells gave rise to proliferative cell lesions that resembled early stages of prostate cancer. From these lesions, Pde4d, Klhl13, Braf, and Nras were identified as CISs. Moreover, Pde4d was shown to be a pro-proliferative factor in human prostate cancer cell lines and to be overexpressed in human prostate cancers. SB mutagenesis in Schwann cells gave rise to all stages of Schwann cell tumorigenesis including benign neurofibromas, plexiform neurofibromas, and aggressive MPNSTs. From these tumors, hundreds of CISs were identified including known tumor suppressors Nf1, Pten, and Cav1. Experiments with loss of Cav1 demonstrated cooperativity with EGFR overexpression in vivo for Schwann cell tumorigenesis.
Investigation of the function and control of Dia2, a regulator of genomic stability in budding yeast.
(2009-11) Kile, Andrew Craig
Maintenance of genomic integrity can be particularly challenged during DNA replication, which is critical for cellular viability and proliferation. Cancer cells exhibit loss of genomic integrity, thus it is critical to understand the pathways involved in genome maintenance. We have identified the F-box protein Dia2 as a novel and previously unappreciated mediator of genome stability. F-box proteins are substrate specificity subunits of SCF ubiquitin ligases for ubiquitin mediated proteolysis, although most remain uncharacterized in their function or targets. Deletion of the DIA2 gene in Saccharomyces cerevisiae leads to genomic integrity defects, and the Dia2 protein associates with chromatin and origins of replication, indicating it performs a chromatin-associated role in DNA replication. Interestingly, the Yra1 protein was identified to physically interact with Dia2 and promotes Dia2 binding to replication origins yet is not a proteolytic substrate of SCF-Dia2. The Dia2 protein itself is subject to proteolysis, but is stabilized by the activation of the replication checkpoint and this suggests it plays a role during periods of replication stress and DNA damage during S phase. Surprisingly, Dia2 turnover is not controlled by an autocatalytic mechanism involving its F-box domain, but instead relies on a region upstream of its F-box that controls both its stability and nuclear localization. Replication checkpoint activation leads to inhibition of late-firing origins, stabilization of replication forks, as well as stabilization of the Dia2 protein. Our observations indicate that SCF-Dia2 activity performs ubiquitin ligase activity at one or both of these sites that are regulated by the checkpoint. These studies establish a novel link between DNA replication and genomic integrity to the SCF ubiquitin ligase via Dia2.
Minisatellites in meiosis: crossover regulation and stability of repetitive DNA.
(2012-04) LeClere, Andrea Ruth
The minisatellite associated with the human HRAS1 proto-oncogene has an enhancer effect on HRAS1 expression. Rare minisatellite alleles have stronger enhancer activity and are frequently found in primary tumors of cancer patients. Rare alleles derive from a common allele that has undergone an alteration in length; alterations occur primarily during the process of meiotic recombination. Identifying factors that regulate minisatellite stability during meiosis is important to our understanding of the initiation and predisposition of minisatellite-associated human diseases. This is the focus of the Kirkpatrick lab using a minisatellite model system in yeast Saccharomyces cerevisiae where an HRAS1 minisatellite allele has been inserted into the promoter region upstream of the HIS4 locus on chromosome III. In this study, we identified CSM3, TOF1, and MRC1 as factors that contribute to meiotic minisatellite alterations. We also uncovered a novel recombination phenotype associated with the HRAS1 minisatellite in MSH4 and MSH5 mutants. Both of these projects have broader implications on our understanding of factors that regulate minisatellite alterations during meiosis. We present models based on our data and previously published research to explain the observed phenotypes.

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