Department: Molecular Genetics ![[clear]](close-x.png "Remove this limiter")
61 matches in the database.
These are records: 1 - 30.
[1] [2] [3]
1.
Arduini, Brigitte L.
Sublineage-specific cues required for early and later neural crest development in the Zebrafish, Danio Rerio.
Degree: PhD, Molecular Genetics, 2005, Ohio State University
► The neural crest (NC) of vertebrate animals gives rise to many derivatives,…
(more)
▼ The neural crest (NC) of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The generation of NC-derived cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. Zebrafish trpm7/touchtone, endzone, and foxd3/sympathetic mutation1 are discrete loci required by subsets of neural crest derivatives. Severe mutant alleles of the divalent cation channel gene trpm7 are lethal and cell-autonomously cause reductions in the number and size of crest-derived melanophores, while sparing other NC lineages. The deficit in cell numbers can be accounted for at least in part by cell death of melanophore precursors. Pleiotropic effects in non-crest derived tissues, including altered order of bone ossification and kidney dysfunction, are observed in homozygotes for semi-viable alleles. Mutations in endzone affect all three pigment cell lineages found in zebrafish. Normally large and stellate, melanophores and xanthophores take on a rounded, punctate appearance in these mutants. Iridiphores are also reduced in size. These three cell types appear to be similarly reduced in numbers in endzone mutant embryos. While neuronal, glial and ectomesenchymal derivatives of the NC appear to be normal in endzone homozygotes, the non-crest-derived pigmented retinal epithelium is developmentally delayed, pointing to pleiotropism for these mutations, as well. Both trpm7 and endzone act relatively late during chromatophore development; accordingly molecular analyses reveal no defects in the early NC cell populations of these mutants. foxd3sym1 affects multiple derivatives within the NC. FoxD3 is required for sympathetic and sensory neuron development, but appears to be dispensable for chromatophore lineages. Anterior elements of the craniofacial skeleton are reduced, and posterior elements are missing, indicating a role for foxd3 in axial patterning of the pharyngeal arches. Expression of critical transcription factors in early crest cells and crest migration are both abrogated, although the premigratory NC population appears to be induced normally. Simultaneous abrogation of Foxd3 and Tfap2a function leads to loss of all chromatophores and most craniofacial cartilages. Collectively, these data suggest that subpopulations with distinct genetic requirements exist within the early neural crest and its later sublineages.
Advisors/Committee Members: Henion, Paul D.
Keywords: zebrafish; neural crest; cell fate specification; differentiation; sublineage
More Like This
2.
Armstrong, Kristin R.
Multiple Mechanisms Contribute to Regulation of Gene Expression in the C. elegans Excretory System.
Degree: PhD, Molecular Genetics, 2008, Ohio State University
► Gene regulation is controlled by multiple mechanisms that function at levels ranging…
(more)
▼ Gene regulation is controlled by multiple mechanisms that function at levels ranging from genome-wide to gene-specific. Understanding the mechanisms of gene regulation at all levels is important for discerning proper organ development and function as well as interpreting the changes that occur in mutant organs, which lead to defects and diseases. To better understand the mechanisms functioning at different levels of gene regulation in organ development and function, I studied the role of the environment, chromatin remodeling, and gene-specific transcriptional regulation on the C. elegans excretory system, which is similar to mammalian kidneys.Class III POU-homeobox transcription factors are expressed in the renal organs of species from C. elegans to mammals. However, the functional role for these factors in renal organs is not well characterized. In this dissertation, I studied the role of the C. elegans POU-III transcription factor, CEH-6, in the excretory cell to address this question. I performed molecular and biochemical studies, which show CEH-6 regulates a subset of genes expressed in the excretory cell. This work suggests that a role for POU-III factors in renal organs may be to modulate the expression of functionally related genes. Chromatin structure also influences the accessibility of transcription factors to target genes, and thereby is important for gene expression. In this study, I show LEX-1 and TAM-1 function to attenuate transgene silencing in somatic tissue by acting regionally as part of a complex that broadly regulates expression of genes in repetitive DNA. Additionally, environmental cues can play a role in regulating gene expression. Previous work by other labs has shown dauer larvae exhibit an increased osmotic tolerance, suggesting the excretory system may be altered. I was interested in understanding the biological changes responsible so I characterized the excretory system anatomy in dauer larvae. I found an altered morphology and the positional alignment of the excretory duct cell and excretory cell in dauer larvae, which may be related to the increased osmotic tolerance. Together, the results from these studies support the idea that organisms integrate information from multiple levels and mechanisms of regulation in order to direct their development and function.
Advisors/Committee Members: Chamberlin, Helen.
Subjects: Anatomy and physiology; Biology; Cellular biology; Genetics; Molecular biology
Keywords: excretory system; osmoregulation; excretory cell, transcriptional regulation; POU-III homeobox; chromatin remodeling; synMuvB; heterochromatin; euchromatin; repetitive DNA; gene silencing; environment; dauer; quiescent
More Like This
3.
Bennett, Kristi Lynn.
Methylation in head and neck squamous cell carcinoma.
Degree: PhD, Molecular Genetics, 2007, Ohio State University
► In order to improve treatment and facilitate timely detection in Head and…
(more)
▼ In order to improve treatment and facilitate timely detection in Head and Neck Squamous Cell Carcinoma (HNSCC), elucidation of early detection markers is crucial. DNA methylation markers are advantageous, because DNA methylation is an early event. Following an overview on methylation in HNSCC in Chapter 1, we describe a genomewide screen using Restriction Landmark Genomic Scanning (RLGS) in Chapter 2. This analysis found a set of potential tumor suppressor genes that are commonly methylated. Not all relevant candidates are detected by RLGS because of limitations of the assay. In Chapter 3, a candidate gene approach identified C/CAAT enhancer binding protein alpha (C/EBPα), a gene previously shown to exhibit tumor suppressor activity in acute myeloid leukemia (AML) and lung cancer, as a gene of interest. C/EBPα tumor suppressor activity in lung cancer has previously been shown to be downregulated by epigenetic mechanisms. More recently, this gene has been found to be downregulated in HNSCC. This prompted investigation into the involvement of epigenetics in downregulating C/EBPα in HNSCC. It was revealed that C/EBPα is downregulated in HNSCC by loss of heterozygosity (LOH) and upstream DNA methylation. Also, C/EBPα overexpression in a HNSCC cell line (SCC22B) revealed its ability to provide tumor suppressor activity in HNSCC in vitro and in vivo. Upstream methylation of C/EBPα correlates with decreased expression in HNSCC. In Chapter 4, investigation of previously unstudied AP2α binding sites within the upstream methylated region demonstrated that AP2α suppresses C/EBPα promoter activity and protein expression. Methylation analysis of the upstream C/EBPα̣ sequence after AP2α downregulation revealed decreased methylation, suggesting that AP2α binding may preceed and facilitate methylation and stable silencing of the gene. Finally, in Chapter 5 we discuss the relevance of the findings in the preceeding chapters and the future direction of this body of work.
Advisors/Committee Members: Plass, Christoph.
Subjects: Biology, Genetics
Keywords: Methylation; HNSCC; C/EBP Alpha; EBF3; SLC5A8; FUSSEL18; IRX1; SEPT9; AP2 Alpha; Transcriptional Regulation
More Like This
4.
Brena, Romulo Martin.
Aberrant DNA methylation in human non-small cell lung cancer.
Degree: PhD, Molecular Genetics, 2007, Ohio State University
► Lung cancer is the leading cause of cancer-related death worldwide. Given its…
(more)
▼ Lung cancer is the leading cause of cancer-related death worldwide. Given its impact on human health, extensive research is being conducted in an effort to reduce the global lung cancer death toll. Specifically, much interest has been placed on the development of biomarkers and the discovery of novel prognostic factors. Over the past 2 decades it has become evident that the cancer genome is not only affected by genetic abnormalities, such as mutations, deletions and chromosomal rearrangements, but also by epigenetic changes which, together, contribute to the deregulation of transcription profiles. Epigenetic changes are defined as heritable lesions to the DNA affecting gene expression without altering the primary DNA sequence. These lesions typically involve a genomewide reduction in 5-methylcytosine, increased DNA methylation in gene promoter sequences and substitutions in histone tail modifications. Epigenetic changes have been shown to interact with one another, resulting in genomic instability, silencing of tumor suppressor genes, activation of oncogenes and derepression of transposable elements. As opposed to the irreversible nature of genetic lesions, epigenetic lesions can be reversed. Because of their reversibility, epigenetic alterations have become an attractive target for new therapies, which has resulted in the development of new anticancer compounds, several of which are currently in clinical trials. The field of epigenetics has expanded considerably since its inception. Integrative research approaches aimed at elucidating the contribution of genetic and epigenetic alterations to the tumorigenic process are being undertaken worldwide, generating promising results. In hopes of furthering the body of knowledge currently afforded by the epigenetics field, the work presented in this thesis is focus primarily on unraveling the role of DNA methylation in the diagnosis, etiology and potential treatment of lung cancer.
Advisors/Committee Members: Plass, Christoph.
Subjects: Biology, Genetics
Keywords: Lung Cancer, OLIG1, DNA methylation
More Like This
5.
Budovskaya, Yelena V.
An Evaluationary Proteomics Approach for the Identification of Substrates of the Camp-Dependent Protein Kinase in Saccharomyces Cerevisiae.
Degree: PhD, Molecular Genetics, 2005, Ohio State University
► Eukaryotic cells utilize a network of signal transduction pathways to sense their…
(more)
▼ Eukaryotic cells utilize a network of signal transduction pathways to sense their environment and control their growth and proliferation. Protein kinases are a large group of enzymes that coordinate responses to extracellular and intracellular stimuli via phosphorylation of specific downstream targets. In S. cerevisiae , growth is controlled, in part, by the Ras signaling pathway via the cAMP-dependent protein kinase, PKA. PKA is a serine/threonine-specific protein kinase that has been shown to regulate any aspects of cell growth and metabolism in this budding yeast and other eukaryotes. Unfortunately, finding protein kinase substrates by conventional methods is a difficult and time-consuming task. As a result, few targets of any given protein kinase are known. To simplify this task, we developed an evolutionary proteomics strategy for the identification of PKA substrates in S. cerevisiae and related yeast species. This evolutionary proteomics approach is sequenced-based and takes advantage of the fact that most PKA substrates contain the consensus sequence, R-R-x-S/T-B. In this consensus, “x” refers to any amino acid, “B” to hydrophobic residues and “S” or “T” to the site of phosphorylation. The general approach consists of two basic steps. In the first, we identified all of the proteins in the S. cerevisiae proteome that contain this PKA target consensus sequence. In the second, we asked whether these potential target sites are conserved in the orthologous proteins present in other budding yeast species. For this latter step, we used the recently released genome sequences of six different yeast, including five Saccharomyces species and Candida albicans. The underlying premise of this approach is that PKA sites important for general aspects of cell biology are more likely to be conserved across these evolutionary distances. We are presently testing this basic premise with a small number of proteins predicted to be physiologically relevant PKA substrates. In this thesis, I will discuss my recent work with one of these potential targets, Atg1p. Atg1p is a key regulator of autophagy, a membrane trafficking pathway that is responsible for much of the protein and organelle turnover occurring during periods of nutrient depravation. This catabolic pathway is highly conserved and Atg1p homologs exist in essentially all eukaryotic organisms. We have found that the S. cerevisiae Atg1p contains two PKA consensus sites that have been conserved in all budding yeast and C. albicans. My work has shown that Ras/PKA signaling activity does indeed control autophagy in vivo and that Atg1p is phosphorylated at the two predicted sites by PKA. PKA phosphorylation of Atg1p does not effect its protein kinase activity, but appears instead negatively regulate the association of Atg1p with the pre-autophagosomal structure (PAS). We hypothesize that this regulation might prevent Atg1p from reaching specific substrates that are important for the autophagy process.
Advisors/Committee Members: Herman, Paul K.
Subjects: Biology, Molecular
Keywords: PKA; Autophagy; Ras; protein kinases; substrates; Atg1
More Like This
6.
Butchar, Jonathan P.
Microarray analysis of drosophila EGF receptor signaling and cell line expression profiles.
Degree: PhD, Molecular Genetics, 2006, Ohio State University
► Despite the enormous advances in our understanding of the Drosophila Epidermal Growth…
(more)
▼ Despite the enormous advances in our understanding of the Drosophila Epidermal Growth Factor Receptor signaling (dEgfr), it is evident that there are targets of this pathway that remain to be discovered. Here, I employed a microarray-based approach to identify previously undiscovered dEgfr targets in the 3rd -instar wing imaginal disc. Candidate genes were verified in vivo and five were analyzed further. Based on in situ hybridization and functional data, I further hypothesized that one gene, Sulf1, acts in a negative feedback loop to regulate the dEgfr pathway. Genetic interaction crosses confirmed this, and showed that Sulf1 also negatively influences the Notch pathway. Tissue culture experiments using Drosophila cells have been performed for approximately the past 45 years (Brooks and Kurtti, 1971). Despite these decades of research little is known about their transcriptional profiles. Further, it is not yet known exactly which genes are responsible for their taking on an immortalized proliferative state rather than differentiating into their final cell types. To address these issues, I have compared Drosophila cell line groups against a pool of imaginal disc and embryonic datasets. This yielded a list of genes which may be involved with generation and / or maintenance of immortalization. In addition, class prediction and hierarchical clustering routines were used to rapidly differentiate cell lines from one another and to classify a newly created cell line. Results were exported into a database and have been made accessible through a web server.
Advisors/Committee Members: Simcox, Amanda A.
Keywords: EGF receptor; microarrays; Drosophila melanogaster; cell lines
More Like This
7.
Carmichael, Jennifer Rose.
The Host-pathogen Relationship in Rickettsia: Epidemiological Analysis of RMSF in Ohio and a Comparative Molecular Analysis of Four vir genes.
Degree: PhD, Molecular Genetics, 2008, Ohio State University
► Members of the vector-borne bacterial genus Rickettsia represent an emerging infectious disease…
(more)
▼ Members of the vector-borne bacterial genus Rickettsia represent an emerging infectious disease threat and have continually been implicated in epidemics worldwide. It is of vital importance to understand the geographical distribution of disease and rickettsial-infected arthropods vectors. In addition, understanding the dynamics of the relationship between rickettsiae and their arthropod hosts will help aid in identifying important factors for virulence. Dermacentor variabilis dog ticks are the main vector in the eastern United States for Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever. The frequency of rickettsial-infected ticks and their geographical location in Ohio over the last twenty years was analyzed. The frequency of rickettsial species was found to remain relatively constant (about 20%), but the incidence of R. rickettsii has increased from 6 to 16%. Also, the geographic distribution of rickettsial-positive ticks has expanded, corresponding to a rise of RMSF in these new areas. Type IV secretion system genes, like the vir group, are important for pathogenicity in many pathogens, but have not been analyzed in Rickettsia. Four vir genes, virB8, virB11, virB4, and virD4 were analyzed in Rickettsia amblyommii infected Amblyomma americanum Lone Star ticks from across the Northeast United States. Results showed that important protein motifs are mutated or absent. This evidence suggests the vir genes may not be functional in Rickettsia or they have evolved a novel role. In addition, analysis of virD4 showed that isolates were most similar to Rickettsia conorii and R. rickettsii, two highly pathogenic species. To determine if these genes are important for virulence, a novel qPCR approach was designed to compare the RNA expression in R. amblyommii-infected A. americanum tick and Vero cells. Here, expression of virB8 and virD4 was increased in Vero cells, while virB4 and virB11 had decreased expression. Since rickettsiae cause disease in humans, increases in expression levels in the mammalian Vero cells may be important for pathogenicity. Understanding all levels of disease, from basic biology and epidemiology, analysis of potential virulence genes, and functional analysis via RNA expression, contributes information that leads us closer to determining the process of rickettsial pathogenicity.
Advisors/Committee Members: Fuerst, Paul.
Subjects: Molecular biology
Keywords: Rickettsia; ticks; Dermacentor; Amblyomma; vir gene; host-pathogen; , qPCR; R. amblyommii
More Like This
8.
Carver, Laura.
Regulation of Slit-Robo Signaling by Commissureless and Comm Family Members.
Degree: PhD, Molecular Genetics, 2011, Ohio State University
► The field of axon guidance is concerned with deciphering how axons use…
(more)
▼ The field of axon guidance is concerned with deciphering how axons use temporal and spatial cues to pattern a precise trajectory. We use the fruit fly Drosophila melanogaster to study embryonic pathway decisions that are fundamental in connecting the bilaterally symmetrical halves of the nervous system at the ventral midline. At the midline choice point, axons make a binary decision regarding whether or not to cross to the opposite side. One signaling pathway controlling midline crossing is Slit-Roundabout repulsive signaling, whereby axons expressing the Roundabout (Robo) receptor bind the midline-expressed chemorepellant ligand, Slit, and sense the midline as inhibitory. Expression of Commissureless (Comm)—and its post-translational re-localization of Robo—effectively silences Slit-Robo signaling, allowing axons to cross the midline. In addition to comm, there exist two other Comm family members: comm2 and comm3. We utilized cell culture and in vivo approaches to learn about comm2, whose spatiotemporal expression is similar to comm during embryogenesis. Like Comm, Comm2 re-localized full-length Robo in S2-based cell culture assays but required sequences in Robo’s extracellular (EC) and/or cytoplasmic (cyto) domain not required by Comm. Additionally, deletion of Comm2’s EC domain abrogated its Robo re-localization ability. In flies, both molecularly-defined deletion of Comm2 and Comm2 overexpression in wildtype had subtle phenotypes, but its expression in comm and comm,comm2 deletion backgrounds revealed a positive role in midline crossing. Interestingly, comparison of Comm2 expression in backgrounds deleted for comm, comm2, or both, revealed a possible inhibitory role for Comm2 at the midline. We also created a series of chimeric fusion proteins among Comm family members. Analysis of these constructs supports an unexpected role for the Comm EC domain in regulating Robo. In the S2 cell culture assay, as little as the Comm EC domain was capable of transforming otherwise non-functional Comm2 and Comm3 transmembrane (TM) and cytoplasmic domains into Robo re-localizing proteins. This affect translated to Comm-like functionality when tested in vivo as well. Furthermore, although full-length Comm2 induced Robo re-localization in cell culture, the presence of its domains in various fusion proteins was unable to promote Robo re-localization. We also focused on Comm3, which has no Comm-like functionality, despite having a conserved TM domain and PY motifs. Its EC domain is not inhibitory to Robo re-localization; rather, it may lack the positive sequences present in Comm, making it a good tool to use for understanding Comm and Comm2 function. Its TM/cyto domains are, however, able to function when combined with a Comm EC domain. Finally, we investigated the mechanism by which Comm regulates Robo, with a focus on ubiquitination of Comm and/or Robo. In contrast to other publications, we did not find a necessary role for Comm ubiquitination in Comm-mediated Robo re-localization in S2 cells or in vivo, as it behaved like wildtype Comm in both environments. On the other hand, in S2 cells, Comm less efficiently re-localized a short, lysine-free Robo construct, but some re-localization still occurred. When both were lysine-free, again, some re-localization still occurred, suggesting that ubiquitination may influence the process but is not absolute.
Advisors/Committee Members: Seeger, Mark.
Subjects: Developmental Biology; Genetics; Molecular Biology; Neurobiology
Keywords: Axon guidance; Commissureless; Roundabout; Drosophila melanogaster; neurobiology; molecular biology; genetics; developmental biology
More Like This
9.
Challa, Anil Kumar.
Identification and functional analysis of Zebrafish orthologs of genes.
Degree: PhD, Molecular Genetics, 2003, Ohio State University
► Cell motility and migration are integral to the complex mechanics of animal…
(more)
▼ Cell motility and migration are integral to the complex mechanics of animal development, growth and maintenance. The precise and stereotypical orchestration of cell motility is controlled by a number of molecules, both within cells and in the extracellular environment. The phenomenon of axonal growth cone motility during neural circuit formation is a special case of cell movement, and has been studied extensively over the past several years. A number of receptor-ligand interactions have been described that regulate this process and one example involves the Roundabout (Robo) receptor and its extracellular, secreted ligand Slit. Both of these molecules are evolutionarily conserved from worms to humans and have been shown to play important roles in axon guidance. To gain a better understanding of Robo orthologs in vertebrates, we chose zebrafish as our model system for the many experimental advatages it offers. A PCR-based strategy was employed to find zebrafish orthologs of Robo. Two orthologs ( robo1 and robo3 ) were identified whose expression patterns in the nervous system and non-neural tissues suggested a role in axon guidance and cell motility. Further characterization of robo3 unveiled the presence of two distinct isoforms ( robo3a and robo3b ). The two isoforms exhibited spatially and temporally dynamic gene expression patterns. Functional analysis of robo3 isoforms using an antisense gene 'knock-down' strategy suggested that robo3b functions during gastrulation, whereas robo3a is required for motor axon pathfinding. This study uncovers a novel function of the Robo receptor family in cell movements during gastrulation, complementing an earlier study that suggests a role of zebrafish Slit orthologs in gastrulation. The implication of Robo-Slit signaling in cell movements during gastrulation underscores the conservation of molecular mechanisms in a variety of cell motility phenomena. Cell motility and migration are integral to the complex mechanics of animal development, growth and maintenance. The precise and stereotypical orchestration of cell motility is controlled by a number of molecules, both within cells and in the extracellular environment. The phenomenon of axonal growth cone motility during neural circuit formation is a special case of cell movement, and has been studied extensively over the past several years. A number of receptor-ligand interactions have been described that regulate this process and one example involves the Roundabout (Robo) receptor and its extracellular, secreted ligand Slit. Both of these molecules are evolutionarily conserved from worms to humans and have been shown to play important roles in axon guidance. To gain a better understanding of Robo orthologs in vertebrates, we chose zebrafish as our model system for the many experimental advatages it offers. A PCR-based strategy was employed to find zebrafish orthologs of Robo. Two orthologs ( robo1 and robo3 ) were identified whose expression patterns in the nervous system and non-neural tissues suggested a role in axon guidance and cell motility. Further characterization of robo3 unveiled the presence of two distinct isoforms ( robo3a and robo3b ). The two isoforms exhibited spatially and temporally dynamic gene expression patterns. Functional analysis of robo3 isoforms using an antisense gene 'knock-down' strategy suggested that robo3b functions during gastrulation, whereas robo3a is required for motor axon pathfinding. This study uncovers a novel function of the Robo receptor family in cell movements during gastrulation, complementing an earlier study that suggests a role of zebrafish Slit orthologs in gastrulation. The implication of Robo-Slit signaling in cell movements during gastrulation underscores the conservation of molecular mechanisms in a variety of cell motility phenomena.
Advisors/Committee Members: Seeger, Mark A.
Keywords: Zebrafish, Roundabout, Slit, Axon Guidance, Gastrulation, Cell motility
More Like This
10.
Chen, Shih-Shih.
Transcriptional Silencing Of Foxd3 Is An Early Event Mediating Epigenetic Silencing In Tcl1 Positive Chronic Lymphocytic Leukemia.
Degree: PhD, Molecular Genetics, 2008, Ohio State University
► Chronic lymphocytic leukemia (CLL) is one of the most common types of…
(more)
▼ Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults; recent findings in CLL diagnosis, mechanism of pathogenesis and therapy options are reviewed in in the introductory Chapter 1. Early stages in the development of CLL have not been explored mainly due to the inability to study normal B-cells in route to transformation. Over-expression of human TCL1, a known CLL oncogene, in murine B-cells leads to the development of mature CD19+/CD5+/IgM+ clonal leukemia with a similar disease phenotype seen in human CLL. In Chapter 2, we used this TCL1 murine model of CLL and corresponding human CLL samples in a cross-species Epigenomics approach to address the timing and relevance of epigenetic events occurring during leukemogenesis. In the TCL1 positive B-cells, aberrant DNA methylation of CpG islands occurred progressively more frequently with age. We found a high concordance of methylated genes in the TCL1 mouse and human CLL. Increased methylation at 9 to 14 months correlated with progressively decreasing levels of microRNAs miR29b/miR29c and increased protein levels of their targets DNMT3A/3B. While the increased protein levels of de novo DNMT transferases can not explain methylated targets in TCL1 mice, in Chapter 3, a predicted Foxd3 binding sequence was much more frequently found in methylated genes (70%) as compared to unmethylated genes (29%). Specifically, Foxd3 promoter methylation is noted at five months, and its expression is silenced within one month as a consequence of a TCL1 activated NFκB repressor complex. Similar silencing of FOXD3 was also found in human CLL. Loss of the FOXD3 transcription factor leads to silencing of downstream targets, chromosome remodeling, promoter specific DNA methylation and thus manifesting its role in early leukemogenesis. During the disease development, one of the Foxd3 target genes, inhibitor of DNA binding protein 4 (Id4) is found methylated at a late stage of leukemogenesis in TCL1 mice. While Id4 has been suggested as a tumor suppressor gene in many types of leukemia including CLL, double or hetero knockout Id4 mice do not develop leukemia. To elucidate the role of late-stage methylated genes in CLL development, in Chapter 4, the outcome of loss of Id4 was further investigated using genetically modified mice. In the presence of the heterozygous TCL1 transgene, the mice with haploid loss of Id4 exhibited elevated white blood cells counts, accelerated death accompanied with enlarged spleens and B cells infiltrated secondary organs; while the same background TCL1 mice tend to have slower disease progression. Non-transformed B-lymphocytes from wild-type as opposed to Id4 haplotype deficiency were more resistant to dexamethasone therapy. Overall, our findings suggest aberrant DNA methylation as an early and accumulated event during CLL disease development, the loss of targets might be required for disease progression in both mouse and human CLL-cells. Finally, in Chapter 5 we discuss the relevance of the findings in the preceding chapters and the future direction of this body of work to understanding the pathogenesis of CLL.
Advisors/Committee Members: Byrd, John.
Subjects: Biomedical research
Keywords: TCL1, CLL, FOXD3, DNA methylation
More Like This
11.
Cho, I-Ming.
Characterizing the Role of Ribosomal Protein L7Ae in Archaeal RNase P Catalysis and Exploring the Use of Archaeal RNase P as a Functional Genomics Tool.
Degree: PhD, Molecular Genetics, 2010, Ohio State University
► RNase P is an essential ribonucleoprotein (RNP) complex that catalyzes 5’-maturation of…
(more)
▼ RNase P is an essential ribonucleoprotein (RNP) complex that catalyzes 5’-maturation of tRNAs. It is composed of an RNase P RNA (RPR), the catalytic moiety, and a variable number of RNase P protein (RPP) subunits: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Towards dissecting protein-aided RNA catalysis, our laboratory has successfully reconstituted functional archaeal RNase P holoenzymes by assembling the RPR and four known RPPs. However, since the in vitro reconstituted holoenzymes exhibit lower specific activity (kcat/Km) and optimal temperature for precursor-tRNA (pre-tRNA) cleavage than the native holoenzyme, it is clear that the archaeal RPPs identified so far do not represent the complete suite present in the native version. In this regard, I investigated and demonstrated that ribosomal protein L7Ae, a human RPP38 homolog, is a true component of RNase P from Methanococcus maripaludis (Mma), a mesophilic archaeon. Western analysis of partially purified native Mma RNase P revealed that L7Ae co-elutes with RNase P activity. Addition of recombinant Mma L7Ae to the Mma RPR and four known RPPs in vitro increases the kcat/Km (by 360-fold) and the optimal temperature to that observed with the native holoenzyme. L7Ae is known to bind kink-turns in RNAs. We identified a kink-turn-like motif in the Mma RPR as a binding site of L7Ae by footprinting and mutagenesis studies. The stimulatory effect of Mma L7Ae on RNase P activity is dramatically reduced upon mutating either the conserved nucleotides in this putative kink-turn or conserved amino acids in L7Ae. Thus, L7Ae represents an interesting example of a cellular protein associated with tRNA processing (RNase P), RNA modification (snoRNPs) and translation (ribosomes). Due to its catalytic RNA moiety and essentiality in all cells, RNase P has been customized for various applications. In addition to pre-tRNA cleavage, bacterial and eukaryal RNase P can also cleave model substrates built from two RNAs. For instance, an RNA termed external guide sequence (EGS) could be designed to form a bipartite pre-tRNA-like molecule that is recognized and cleaved by RNase P. This approach has been used to cleave and inactivate cellular target RNAs by expressing the desired EGSs inside cells. We examined the feasibility of using archaeal RNase P for degrading cellular RNAs and thereby down-regulating their function. I demonstrate that bipartite substrates, comprised of a target RNA and EGS, are efficiently cleaved by in vitro reconstituted archaeal RNase P and that EGSs are recycled, indicating that EGSs can provide multiple rounds of substrate recognition and cleavage. Additionally, our results suggest that higher assay temperatures and the preferential binding of EGS to the substrate (compared to the product) contribute to EGS recycling. Together, these studies provide new insights into interactions between an RNA-binding protein and a specific RNA structural motif that is critical for archaeal RNase P catalysis. Moreover, we demonstrate EGS- and archaeal RNase P-mediated RNA cleavage in vitro. Results from these studies also furnish critical insights into factors that will influence the success of this RNA-mediated RNA-cleavage strategy as a gene knock-down tool in archaea.
Advisors/Committee Members: Gopalan, Venkat.
Subjects: Biochemistry
Keywords: RNase P, ribonucleoprotein, tRNA, kink turn (K turn), L7Ae, external guide sequence
More Like This
12.
Chong, Jean-Leon.
Functional characterization of Ded1p, a DExD/H box RNA helicase, in saccharomyces cerevisiae.
Degree: PhD, Molecular Genetics, 2004, Ohio State University
► The yeast DED1 protein (Ded1p) belongs to an evolutionarily conserved DExD/H-box protein…
(more)
▼ The yeast DED1 protein (Ded1p) belongs to an evolutionarily conserved DExD/H-box protein family. Members of this family are involved in essentially all aspects of RNA metabolism, including pre-mRNA splicing, ribosomal biogenesis, RNA transport, and translation. They are often regarded as RNA helicases, or RNA unwindases, because some of them can couple ATP hydrolysis to unwinding short RNA duplexes in vitro. However, recent results suggest that, in vivo, DExD/H box proteins may dissociate specific RNA-binding proteins to remodel ribonucleoprotein complexes. Our lab previously found that Ded1p is a novel essential translation factor. However, Ded1p’s mechanistic role in translation remains to be defined.To examine Ded1p’s role further, I sought to identify its interacting proteins. In the first part of this dissertation (Chapter 1 to 4), I describe the unexpectedly finding that Ded1p binds to yeast L-A virus particles and accelerates the rate of L-A’s negative-strand RNA synthesis in vitro. Viruses are intracellular parasites that must use the host machinery to multiply, because their genomes are very small. Thus, identification of the host factors that perform essential functions in viral replication is of crucial importance to the understanding of virus-host interactions. The findings in this dissertation and the fact that Ded1p is also required for translating the brome mosaic virus RNA2 in yeast thus raise an intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses including the human hepatitis C virus.In the second part of this dissertation (Chapter 5), I show that Ded1p is associated with active spliceosomes and inactivation of Ded1p affects spliceosome formation. Moreover, data from our lab demonstrates that introns accumulate in ded1 mutant strains after being shifted to non-permissive temperature. These findings indicate that Ded1p is also involved in pre-mRNA splicing. A growing body of evidence has suggested that many proteins can function in different nuclear processes in gene expression, implying a close inter-dependent relationship among these processes. The observations in this dissertation and the fact that Ded1p is a translation factor suggest a possible intimate coordination in gene expression between the nucleus and the cytoplasm.
Advisors/Committee Members: Chang, Tien-Hsien.
Subjects: Biology, Genetics
Keywords: DED1P; L-A VIRUS
More Like This
14.
Crary, Monica J.
Genetic Variability and its Relationship to Acanthamoeba Pathogenesis.
Degree: PhD, Molecular Genetics, 2012, Ohio State University
► Acanthamoeba is a pathogenic protist that causes a sight-threatening eye infection, Acanthamoeba…
(more)
▼ Acanthamoeba is a pathogenic protist that causes a sight-threatening eye infection, Acanthamoeba keratitis (AK). The central focus of this research is to analyze the genetic relationships between Acanthamoeba isolates and how that contributes to pathogenesis. Acanthamoeba is a diverse genus with more than 18 genotypes based on the 18S ribosomal RNA gene. DNA barcoding has suggested that the mitochondrial cytochrome oxidase c subunit 1 (COI) gene can be accurately used to identify a eukaryotic organism. As part of understanding more about the phylogenetic structure of the genus of Acanthamoeba, representative isolates of most of the genotypes were analyzed using their COI genes. The phylogenetic relationships based on the COI gene were nearly identical to those produced using the 18S rRNA gene. This allows COI to be used as a reasonable substitute to the 18S rRNA gene. This project found an alternative method of classifying Acanthamoeba and allowed Acanthamoeba phylogenetics to be examined at more than one locus. A majority of this research focused on the ongoing Acanthamoeba keratitis outbreak in Chicago, Illinois since 2002. The dramatic increase of AK cases has occurred following EPA mandated water treatment changes which have been hypothesized to have increased microorganisms in the water system. To understand the phylogenetic structure of Acanthamoeba within this outbreak, a multilocus sequence typing (MLST) protocol was created. This project involved identifying and developing sequencing methods for five highly conserved housekeeping genes in Acanthamoeba. Sequences from these genes were used to determine the degree of variability amongst Acanthamoeba as well as to further our understanding of sub-lineages within the most common genotype, T4. In addition to the Chicago AK review, three surveys were conducted to elucidate the diversity of environmental Acanthamoeba from Chicago, Sonora, Mexico and on an Ohio farm. These surveys combined with the MLST study demonstrate how diverse the genus of Acanthamoeba is, regardless of source. Acanthamoeba are also known to contain potentially pathogenic endosymbionts including Legionella and Pseudomonas. The presence of endosymbionts has been shown to increase the pathogenicity of Acanthamoeba. To determine if endosymbionts in Acanthamoeba could contribute to the Chicago outbreak, Acanthamoeba was analyzed for the presence of Legionella and Pseudomonas. More than 50% of Acanthamoeba from Chicago contained one or both bacterial genera and these bacteria were located intracellularly in Acanthamoeba. We hypothesize that increases in microorganisms in the Chicago water systems increased the opportunity for Acanthamoeba to associate with pathogenic bacteria. Future research will examine the mechanism that these bacteria use to increase Acanthamoeba’s virulence as well as monitoring if diseases associated with these bacteria are also increasing. Together, these projects are meant to further our understanding on the diversity of the genus Acanthamoeba and the underlying factors of Acanthamoeba’s virulence.
Advisors/Committee Members: Fuerst, Paul.
Subjects: Genetics
Keywords: Acanthamoeba; Legionella; Pseudomonas; phylogenetics
More Like This
15.
Davies, Jonathan Robert.
Characterization of two nima interacting proteins suggests a link between nima and nuclear membrane fission.
Degree: PhD, Molecular Genetics, 2004, Ohio State University
► In the filamentous fungus Aspergillus nidulans, the NIMA kinase is required along…
(more)
▼ In the filamentous fungus Aspergillus nidulans, the NIMA kinase is required along with CDK1/cyclinB for mitotic entry. The essential function of NIMA in A. nidulans and the growing recognition of its importance in other eukaryotes, means that the study of NIMA function should reveal unique insights into cell cycle regulation amongst a broad range of organisms. I describe here the characterization of TINC and TIND, two NIMA interacting proteins identified in a yeast Two-hybrid screen, and describe the potential novel roles they may play in mitotic regulation. TINC and a related protein in A. nidulans, An-HETC, are highly similar to proteins conserved in filamentous fungi. Strains which lack both tinC and An-hetC are viable, but do display osmotic and cold sensitivity. Characterization of TINC suggests that it is involved in mitotic regulation. First, TINC is present in the nucleus during mitosis. Second, TINC interacts with NIMA in a phosphorylation state dependant manner. Third, truncated forms of TINC (ΔN-TINC) produce cell cycle defects characterized by a defect in nuclear membrane fission in which cells are able to separate DNA but unable to cleave the nuclear envelope. Significantly, ΔN-TINC localizes to membranous bodies which associate with nuclei. Finally, expression of ΔN-TINC promotes premature loss of NIMA from mitotic samples. The second NIMA interacting protein, TIND, is conserved from bacteria through to humans. TIND is predominantly mitochondrial throughout the cell cycle, but work in other organisms suggests that alternate forms of TIND may exist outside the mitochondria. Expression of a form of TIND which lacks the mitochondrial targeting peptide (ΔN-TIND) produces nuclear division and nuclear membrane fission defects similar to those seen for ΔN-TINC. Additionally, ΔN-TIND also produces defective mitotic spindles, which appear monopolar, an effect not seen for ΔN-TINC. The facts that TINC and TIND were isolated as NIMA interacting proteins, TINC interacts with NIMA in A. nidulans, and expression of ΔN-TINC or ΔN-TIND produces specific defects in nuclear membrane fission suggest roles for these proteins in mitotic regulation. Additionally, the finding that NIMA is destabilized in cells displaying mitotic defects suggests a role for NIMA in regulating nuclear membrane fission.
Advisors/Committee Members: Osmani, Stephen A.
Keywords: Aspergillus nidulans; mitosis; NIMA; nuclear membrane fission
More Like This
16.
Deshpande, Rashmi Jayant.
POP-1/CETCF-1 has multiple functions in P ectoblast development.
Degree: PhD, Molecular Genetics, 2005, Ohio State University
► Wnt pathways are highly conserved pathways that regulate key processes such as…
(more)
▼ Wnt pathways are highly conserved pathways that regulate key processes such as cell type specification and migration of cells during animal development. The C. elegans vulva displays symmetry such that the posterior half of the vulva forms a mirror image of the anterior half. We observed that nuclear levels of POP-1 also display similar mirror symmetry in the developing vulva. We are studying the processes that establish this mirror symmetric pattern of POP-1 localization. Because mutations in the putative WNT receptors lin-17/frizzled and lin-18/Ryk were known to cause defects in the posterior half of the vulva, we examined POP-1 localization patterns in these mutants. We observed that POP-1 localization is reversed in the posterior most part of the vulva with respect to wild-type, in these mutants. lin-17/lin-18 double mutants show a more penetrant reversal of POP-1 localization, suggesting that LIN-17 and LIN-18 act in a semi-redundant manner to reorient POP-1 localization in the posterior half of the vulva. We have investigated the mechanisms by which two other P ectoblasts, P11 and P12 are specified. Prior studies have shown that lin-44/Wnt, lin-17/Frizzled and bar-1/canonical b-catenin function in P12 specification. We have obtained additional evidence that a canonical Wnt pathway functions in P12 specification. We have demonstrated that activated forms of BAR-1 (deltaNBAR-1) are sufficient to cause a P11 to P12 transformation. Interestingly, time-course studies with deltaNBAR-1 expression suggest that Wnts may act at the same time as the EGF pathway, suggesting the requirement of two parallel pathways. We predict a function for POP-1 in P12 specification as BAR-1 and b-catenins in other organisms are known to act primarily through TCFs to activate transcription. We found that the pop-1 hypomorphs, hu9 and q645 that should reduce bar-1 activity cause a low rate of P12 to P11 transformations. This supports the hypothesis that POP-1 acts in P12 specification in a BAR-1 dependent manner. Surprisingly, we find that pop-1 RNAi causes a P11 to P12 transformation, revealing a second function of POP-1 in P11 specification. This antagonistic function of POP-1 appears to be independent of BAR-1 as observed by epistasis experiments. We find that multiple Wnts act in P12 specification. lin-44(n1792/Wnt) and egl-20(n585)/Wnt double mutants display an enhanced rate of P12 to P11 transformations. Genetic analysis using other Wnts and Wnt receptors possibly functioning with LIN-44 and LIN-17 in P12 specification will be discussed.
Advisors/Committee Members: Hill, Russell James.
Subjects: Biology, Cell
Keywords: Wnt signaling, canonical, non-canonical mechanisms, POP-1, P ectoblasts
More Like This
17.
Dhungel, Nripesh.
tRNA Splicing Endonuclease: Novel and Essential Function Beyond tRNA Splicing and Subunit interactions.
Degree: PhD, Molecular Genetics, 2012, Ohio State University
► tRNA splicing is a multi-step process in all eukaryotes. In yeast Saccharomyces…
(more)
▼ tRNA splicing is a multi-step process in all eukaryotes. In yeast Saccharomyces cerevisiae and vertebrates, the enzyme catalyzing intron removal from pre-tRNA is a heterotetrameric complex (SEN complex) consisting of four subunits: Sen2, Sen15, Sen34, and Sen54. Although the SEN complex is conserved between yeast and vertebrates, the subcellular location where pre-tRNA splicing occurs is not. In budding yeast, the SEN complex is located at the cytoplasmic surface of mitochondria, whereas in vertebrates, pre-tRNA splicing is nuclear. Two-hybrid analyses have shown that Sen2 interacts with Sen54 and Sen15 interacts with Sen34; however, the exact mechanism of how the heterotetrameric subunits assemble at their endogenous location is unknown. To understand why tRNA splicing occurs at the outer surface of mitochondria in yeast, I engineered yeast to mimic the vertebrate cell biology by expressing nuclear localized tRNA splicing endonuclease subunits and tRNA ligase. Utilizing Northern and RT-PCR analyses, fluorescence in situ hybridization, and a reporter for tRNA aminoacylation, the data demonstrate that all three steps of pre-tRNA splicing; intron removal, ligation, and 2′-phosphate transfer, as well as nuclear export of tRNA and aminoacylation occur efficiently in yeast cells lacking endogenous mitochondrially located tRNA SEN. Therefore, the canonical function for tRNA splicing endonuclease can occur efficiently in the nucleus when yeast cells express nuclear splicing proteins. However, because pre-tRNA splicing in the nucleus fails to complement growth defects of cells with defective endogenous mitochondrially located splicing endonuclease, my studies also show that the yeast SEN complex surprisingly serves a novel and essential function in the cytoplasm that is unrelated to tRNA splicing. The novel function requires all four SEN complex subunits and the catalytic core of the complex. Exploring the novel function, the data show that a subset of pre-rRNAs accumulate when the SEN complex is restricted to the nucleus, indicating that the SEN complex moonlights in rRNA processing. To study interaction and assembly of tRNA SEN subunits in yeast, two sets of plasmids were generated. One set encodes nuclear tRNA SEN tagged with mCherry and the other encodes endogenously located mitochondrial tRNA SEN tagged with GFP. By utilizing the plasmids in co-transformation studies, this study confirms previously published two-hybrid interactions between splicing subunits and also demonstrates a homodimeric interaction between Sen15 subunits in vivo for the first time in yeast, consistent with a recent crystal structure study that predicts Sen15 homodimers in humans. These studies demonstrate for the first time that 1) tRNA splicing can occur in the nucleus of yeast cells that express nuclear splicing proteins but lack functional endogenous mitochondrial SEN, 2) tRNA SEN has a novel and essential function that is unrelated to tRNA splicing, and 3) the novel function of tRNA SEN may be involved in rRNA processing, either directly or indirectly. Taken together, these findings suggest that selection for the subcellular distribution of the SEN complex in yeast may reside not in its canonical, but rather in a novel and essential, activity. Additionally, the study has led to a new method for studying complex protein interactions in vivo, validated by proof-of-principle experiments in addition to possibly uncovering yet unknown Sen15-Sen15 interactions. Sen15-Sen15 interactions are not expected to function in pre-tRNA splicing and therefore the putative dimer may also have another function unrelated to tRNA splicing.
Advisors/Committee Members: Hopper, Anita K.
Subjects: Biology; Cellular Biology; Genetics; Molecular Biology
Keywords: tRNA splicing endonuclease, tRNA ligase, phosphotransferase, rRNA processing, pontocerebellar hypoplasia
More Like This
18.
Egriboz, Onur.
THE MOLECULAR MECHANISMS GOVERNING THE GAL GENE SWITCH OF SACCHAROMYCES CEREVISIAE.
Degree: PhD, Molecular Genetics, 2012, Ohio State University
► Many insights into how genes are regulated in higher organisms including humans…
(more)
▼ Many insights into how genes are regulated in higher organisms including humans have stemmed from over four decades of studies on the Saccharomyces cerevisiae GAL gene switch. Basic mechanisms remain to be elucidated concerning the three principal GAL gene switch proteins, Gal3, Gal80 and Gal4. The transcriptional regulation of the GAL genes is repressed in absence of galactose via Gal80 inhibition of Gal4, a site-specific DNA binding transcriptional activator. Galactose binds to Gal3, converting it to a form that binds to Gal80 and releases Gal4 from the Gal80 inhibition. It was thought that such a mechanism, consistent with the data at the time, could explain the very rapid response to galactose that is a hallmark of this system. According to one previous working model, it was proposed that Gal80 shuttles rapidly between nucleus and strictly cytoplasmic Gal3 sequesters Gal80 in the cytoplasm in response to galactose. That model proposed dissociation of Gal80 from Gal4 in response to galactose. Some previous work led to data against dissociation of Gal80 from Gal4, while other previous work provided evidence for dissociation. Studies initiated in the course of this thesis work aimed at testing the above competing models and discovering how binding of Gal3 to Gal80 alters Gal80 so as to promote Gal4-mediated activation of GAL gene transcription. We first discovered that Gal3 is uniformly distributed between nucleus and cytoplasm. We found that Gal80 does not show rapid nucleo-cytoplasmic shuttling. And we showed by fluorescence recovery after photobleaching that Gal80 did not rapidly shuttle between the two compartments. We also tested the notion that Gal3 binding to Gal80 interferes with Gal80 self-association, and we found that it does. All together, these results led to a new model, where we proposed the initial Gal3-Gal80 interaction occurs within the nucleus and destabilizes the Gal80 dimer or multimer associated with Gal4, causing Gal80 to dissociate from Gal4. We determined that Gal80-Gal4 complex at the promoters has a slow off-rate, and suggest that Gal3 interacts transiently with Gal4-associated Gal80 to promote its dissociation from Gal4. During search for previously proposed NLS and NES sequences we discovered a phospholipid-binding surface of Gal80. We then discovered that Gal80 could associate with biological membranes extracted from yeast extracts, and that Gal4 could associate with membrane-bound Gal80. On the other hand, we found that Gal3 binding to Gal80 causes dissociation of Gal80 from membranes. That is not the case in the Gal4-Gal80 complex, as solved by the same group. Thus, the Gal3 binding to Gal80 would seemingly prevent membrane association of Gal80. In fact, we present evidence for clusters of Gal80 within the nucleus, and such clusters dissipate in response to Gal3 binding to Gal80 (in galactose). Based on observations in this thesis taken together with data from others, we proposed a broader model of the GAL gene switch that incorporates nuclear clustering of Gal80 multimers at the nuclear periphery as a mechanism for maintenance of tight Gal80-mediated repression of the GAL genes in the absence of galactose.
Advisors/Committee Members: Hopper, James.
Subjects: Molecular Biology
Keywords: Galactose cerevisiae GAL Gal4 Gal80 Gal3
More Like This
19.
Festing, Maria H.
Generation of novel conditional and hypomorphic alleles of the Smad2 gene and the effects of Smad2 removal in environments with elevated retinoid signaling.
Degree: PhD, Molecular Genetics, 2007, Ohio State University
► Smad2 is an intracellular mediator of the transforming growth factor beta (TGF-β)…
(more)
▼ Smad2 is an intracellular mediator of the transforming growth factor beta (TGF-β) signaling pathway, and regulates target gene transcription. It has been previously shown that, in the mouse, removal of functional Smad2 results in embryonic lethality due to defects in gastrulation. To circumvent this early lethality, the Cre-loxP system was utilized to generate a Smad2 conditional allele. Cre-mediated recombination results in a deletion allele which phenocopies the previously reported Smad2ΔC null mutation. The hypomorphic allele Smad23loxP was also created that can be maintained as a homozygote. However, Smad23loxP/Δ embryos are lethal and encompass a variety of phenotypes, including craniofacial midline defects. Holoprosencephaly (HPE) comprises a spectrum of craniofacial midline defects and is a congenital cephalic disorder. Many candidate factors have been identified that are associated with HPE in humans and animals, which include TGIF. TGIF is a corepressor of the retinoid signaling pathway, and removal of this repressor results in increased retinoid signaling. TGIF can also function in as a corepressor the transforming growth factor-beta (TGF-β) pathway, interacting with Smad2. Research has indicated that mouse embryos harboring Tgif mutations are more susceptible to teratogenesis induced by exogenous all-trans retinoic acid. Interestingly, embryos harboring either Smad2 null or hypomorphic alleles are also more susceptible to atRA, and frequently display HPE. Embryos that harbor mutations in both Smad2 and Tgif exhibit a partially-penetrant craniofacial phenotype, which encompasses the HPE spectrum. This indicates that retinoid signaling may be involved in these HPE phenotypes. There are many proteins involved in retinoid signaling, which includes both the retinoid signaling cascade and retinol metabolism. Analysis of gene transcription to determine a possible inherent susceptibility to teratogenesis in Smad2 heterozygous embryos indicated that Adh4 and Cyp26A1, which are both involved in retinol metabolism, were reduced in early embryonic stages when forebrain development is beginning. These transcriptional reductions do not by themselves result in lethality, but when combined with any other factor that perturbs retinoid signaling, HPE can result. Finally, as another method to affect retinoid signaling, a Cyp26A1 null allele was bred to the Smad2 heterozygotes. Consistent with affecting retinoid signaling, the Cyp26A1-/-; Smad2ΔE9,10/+ embryos display HPE.
Advisors/Committee Members: Weinstein, Michael B.
Subjects: Biology, Genetics
Keywords: Smad2 null allele; HPE; Retinoid Signaling; TGF-beta signaling
More Like This
20.
Forsthoefel, David J.
A molecular genetic analysis of the role of the Guanine Nucleotide Exchange Factor Trio during Axon Pathfinding in the Embryonic CNS of Drosophila melanogaster.
Degree: PhD, Molecular Genetics, 2005, Ohio State University
► The Drosophila melanogaster embryo is an ideal system in which to study…
(more)
▼ The Drosophila melanogaster embryo is an ideal system in which to study axon guidance, because of the relative simplicity of the nervous system and the evolutionary conservation of the molecules utilized during development. The Abelson cytoplasmic tyrosine kinase regulates actin cytoskeletal dynamics in Drosophila, mice, and humans. In Drosophila, Abl is expressed in the developing central and peripheral nervous systems (CNS and PNS). In a genetic screen for modifiers of the Abl mutant semilethality phenotype, we identified trio, a cytoplasmic guanine nucleotide exchange factor that is also expressed in the CNS and regulates actin dynamics through Rho GTPases. Mutations in Abl and trio interacted genetically, leading to dramatic disruption of axon pathways at the CNS midline. Building upon these initial observations, we analyzed interactions between Abl, trio, and the attractive Netrin receptor frazzled (fra)/Deleted-in-Colorectal-Cancer (DCC). In fra;Abl and fra;trio double mutants, few axons crossed the midline, similar to the phenotype in trio,Abl mutants. Furthermore, mutations in Abl and trio suppressed the inappropriate midline crossover phenotype in embryos expressing the chimeric Robo-Fra receptor, consistent with an in vivo role for these molecules as Fra effectors. Fra bound Abl and Trio in coimmunoprecipitation and GST pulldown experiments, and tyrosine phosphorylation of Fra and Trio was elevated in cultured cells overexpressing Abl. Mutations in enabled (ena), another Abl substrate, suppressed the loss-of-commissure phenotype in fra, Abl, and trio mutants, as well as the Robo-Fra receptor phenotype. Together, these results suggest that Abl and Trio are effectors for multiple attractive receptors at the CNS midline, and that Ena may function during both attractive and repulsive signaling. Finally, a functional analysis of the requirement for Trio’s conserved domains has been initiated. In transgenic rescue and overexpression experiments, TrioGEF1 was required for axon guidance across the CNS midline, while TrioSH3 inhibited midline crossing. Coexpression experiments with the Robo-Fra receptor and assays in other tissues and cultured cells suggest that the conserved N-terminal domain, spectrin-like repeats, and GEF2 domain may modulate GEF1 signaling in specific contexts. Future experiments must elucidate the mechanistic details of cytoskeletal control by Trio and Fra.
Advisors/Committee Members: Seeger, Mark A.
Keywords: Axon Guidance; Actin Cytoskeleton; Drosophila melanogaster CNS midline; Growth Cone Attraction; Abelson tyrosine kinase (Abl); Trio guanine nucleotide exchange factor; GEF; Enabled (Ena); Mena/VASP; Frazzled (Fra); Deleted in Colorectal Cancer (DCC)
More Like This
21.
Hage, Rosemary.
Identification and characterization of YNL187, a novel factor that promotes stable association of the U1 SNRNP with the 5’SS during pre-messenger RNA splicing.
Degree: PhD, Molecular Genetics, 2007, Ohio State University
► This dissertation describes the identification of a novel splicing factor, Ynl187p, and…
(more)
▼ This dissertation describes the identification of a novel splicing factor, Ynl187p, and characterization of its role in precursor-messenger RNA splicing in the budding yeast, Saccharomyces cerevisiae. The discovery that loss of function mutations in YNL187w can bypass the requirement for Prp28p, an otherwise essential DExH/D-box protein and splicing factor, suggests that Ynl187p is a potential target of Prp28p. Because Prp28p is required for dislodging the U1 snRNP from the intron 5' splice site during pre-mRNA splicing, its targets are predicted to promote stable U1 snRNP/5' ss association during 5' ss recognition and commitment complex formation. I show here that Ynl187p is synthetic-lethal with loss of function mutations in a specific subset of factors that stabilize the U1/5' ss interaction, most of which also bypass Prp28p. Deletion of Ynl187p is sufficient to overcome hyperstabilization of the U1 snRNA/5' ss interaction, indicating that it makes a significant contribution to U1/5' ss stability. Cross-linking studies revealed that contacts between specific proteins and the pre-mRNA are diminished in the absence of Ynl187p and that commitment complexes are unstable. Ynl187p localizes primarily to the nucleus and interacts directly with all five major spliceosomal snRNAs, demonstrating that it interacts with the spliceosome, but is not a snRNP-specific component. Taken together, these findings indicate an important role for Ynl187p in 5' ss recognition and in promoting proper assembly of the first commitment complex in splicing.
Advisors/Committee Members: Chang, Tien-Hsien.
Keywords: pre-messenger RNA splicing
More Like This
22.
Hester, Mark.
Utilization of gene knockout approaches in the mouse to elucidate additional functions of smad proteins during mammalian development.
Degree: PhD, Molecular Genetics, 2005, Ohio State University
► Smad proteins are the intracellular mediator proteins for TGF-beta, activin, nodal, and…
(more)
▼ Smad proteins are the intracellular mediator proteins for TGF-beta, activin, nodal, and BMP signaling molecules. Smad1, 5, and 8 belong to a sub-class that become activated by bone morphogenetic proteins (BMPs), which can multimerize with Smad4 (co-Smad), whereby this complex can translocate into the nucleus to modulate gene transcription. Previous research has shown that Smad1 is important in the formation of the allantois, while Smad4 has been shown to be equally important in the development of extraembryonic tissues. To further analyze the BMP-responsive Smads, the murine Smad8 gene was disrupted utilizing the Cre/loxP system. A Smad8 hypomorphic allele was constructed that contains an in-frame deletion of exon three, removing one third of the MH2 domain and a small portion of the linker region. Although these embryos are phenotypically normal, homozygotes of another allele of Smad8 (Smad83loxP) that contains a neomycin cassette within intron 3, phenocopy an embryonic brain defect observed in roughly 22% of Smad1+/- embryos analyzed at E11.5. Specifically, these embryos exhibited a drastic reduction of hindbrain and midbrain structures. To further characterize the functions of Smad proteins during mammalian development, Smad4 was conditionally deleted within the mouse utilizing the FSP-Cre driver strain. Utilizing this strain eliminates expression of Smad4 in fibroblastic, mesenchymal cell types. Mice depleted for Smad4 utilizing this driver strain exhibit multiple phenotypes such as deformed digits, hair follicle dysplasia, and overall immune and health deficiencies. Histological analysis of Smad4 mutant hair follicles show irregular, enlarged hair follicles, dermal cysts, hyperkeratosis, and utriculi. Further analysis has revealed a defect in mutant hair follicles entering the catagen stage of the hair cycle. The catagen stage is characterized by an apoptosis-driven regression phase of the hair cycle that results in the remodeling of the hair follicle. In addition, increased cell proliferation and decreased apoptosis are observed in mutant hair follicles suggesting a mechanism underlying these enlarged hair follicles. Interestingly, certain developmental processes do not require Smad4 in order to relay BMP and TGF-beta signals into the nucleus to regulate gene transcription, but we have shown its critical requirement in the control of hair follicle growth and cycling.
Advisors/Committee Members: Weinstein, Michael.
Subjects: Biology, Molecular
Keywords: Smad1; Smad8; Pax3; hair follicle; follicle; SIP1; Smad83loxP
More Like This
23.
Johnson, Ryan William.
Genetic Regulation of Caenorhabditis Elegans Post-Embryonic Development Involving the Transcription Factors EGL-38, VAB-3, and LIN-14.
Degree: PhD, Molecular Genetics, 2008, Ohio State University
► Sequence-specific transcription factors are crucial to generating the gene expression patterns that…
(more)
▼ Sequence-specific transcription factors are crucial to generating the gene expression patterns that drive the specification, morphogenesis, and physiology of organs and tissues. In order to better understand how organ form and function are orchestrated by transcription factors, we must better understand the genetic inputs and outputs of these critical regulators. In this work, I have utilized C. elegans to characterize the genetic networks and organ functions of three post-embryonically functioning transcription factors.The Pax family of transcription factors is highly conserved across animal species, and controls the development of multiple tissues and organs during development. In C. elegans males, two sensory mating structures, the copulatory spicules and the post-cloacal sensilla, are formed from stereotyped divisions of the two post-embryonic blast cells, B.a and Y.p, respectively. A C. elegans pax-6 transcript, vab-3, is necessary for the development of these sensory structures. Using a green fluorescent protein (GFP)-based vab-3 transcriptional reporter, I found that expression is restricted to the sensory organ lineages of B.a and Y.p. Transcription of vab-3 in the tail region of the worm requires the Abdominal-B homeobox gene, egl-5. Opposing this activation, a transcription factor cascade and a Wnt signaling pathway each act to restrict vab-3 expression to the appropriate cell lineages. Another C. elegans Pax gene, egl-38, is required for the development of the egg-laying system and rectum. However, few EGL-38 target genes are known. Using gene expression microarrays, we cross-referenced microarray data from an inducible EGL-38 strain and two egl-38 mutants that disrupt protein function in a tissue-preferential manner to identify potential tissue-specific EGL-38 target genes. One set of genes from this analysis was validated using GFP reporter transgenes. Most of these genes are expressed in egl-38-dependent tissues, and many display egl-38 dependence. In addition to the identification of target genes, this work revealed enrichments in gene classes that play a role in innate immunity. Consistent with this, we discovered a novel immune function for egl-38. We found that the gene activities of egl-38 and three egl-38-responsive genes from our validation set are associated with increased infection by the pathogenic bacterium M. nematophilum. However, we also show that egl-38 does not impact infection by a different pathogen, S. marcescens. While Pax genes regulate spatial tissue/organ identity, some transcription factors regulate temporal identity. In C. elegans, heterochronic genes function to ensure the precise timing of stage-specific developmental events. I positionally cloned a novel missense allele of the heterochronic transcription factor LIN-14, and revealed a previously undiscovered ability of this protein to solely affect late larval development. lin-14(sa485) hermaphrodites exhibit asynchrony between vulval and gonadal morphogenesis and maturation. Further, lin-14(sa485) preferentially disrupts the timing of vulval cell morphogenesis, but not cell division. I also show that terminal differentiation of a uterine cell type is delayed in lin-14(sa485) mutants.
Advisors/Committee Members: Chamberlin, Helen.
Subjects: Genetics
Keywords: transcription factor; gene expression; pax; heterochronic; post-embryonic development; innate immunity
More Like This
24.
Jowdy, Casey C.
The Regulation of Commissureless in the Embryonic CNS of Drosophila melanogaster.
Degree: PhD, Molecular Genetics, 2010, Ohio State University
► During development the precise wiring of the nervous system is dependent on…
(more)
▼ During development the precise wiring of the nervous system is dependent on the correct repertoire of axon guidance molecules being expressed at the right time and place. In the CNS of D. melanogaster, Comm and Robo play important roles in regulating axons at the midline. To date much has been studied about how Comm regulates Robo, but little is known about how Comm is regulated in the CNS. Comm is dynamically expressed in an off, on, and off again fashion in contralaterally projecting axons and is not expressed in ipsilateral projecting axons. Our hypothesis is the dynamic spatial and temporal regulation of Comm is at the transcriptional level. In our attempt to understand the molecular basis of Comm expression, we have undertaken several approaches to identify the necessary cis-regulatory elements. Experiments using large P[acman] rescue constructs fail to restore proper Comm expression in the CNS, suggesting that the cis-regulatory regions for Comm may be quite large. To further our understanding of these potentially large cis-regulatory regions, we have taken advantage of site-specific recombination using the yeast enzyme FLP recombinase and FLP recognition target (FRT) sites to make molecularly defined deletions. A series of deletions that remove large portions of DNA, as far as 71 kb 5! of Comm and deletions 45 kb 3! of Comm, have been tested with existing comm alleles. Analysis of these deletions using monoclonal antibodies BP102 and 1D4 to reveal axon projections in the CNS show deletions 5! of Comm have a Robo-like phenotype, where axons cross the midline inappropriately. Examination by fluorescent in situ hybridization for Comm transcript at single cell resolution using genetically defined markers to reveal a subset of (ipsilaterally projecting) neurons reveals Comm accumulation in the cell body where Comm is not normally expressed. In a subset of commissurally crossing eagle (Ew) neurons Comm accumulation is detected earlier than normal. In summary, we have identified the 5! region of Comm as crucial for negatively regulating Comm expression in the CNS. In addition to Comm, there are two Comm-like genes in Drosophila, Comm2 and Comm3. Comm3 is expressed too late in embryogenesis to play a role in midline guidance. However, we show that Comm2 is largely co-expressed with Comm in a similar pattern throughout embryogenesis. Unlike Comm, deletion of Comm2 has no detectable BP102 or 1D4 phenotype; a mild Robo-like gain-of-function phenotype is produced when Comm2 is pan-neurally expressed in the CNS. A screen for dominant modifiers of the Netrin deficiency phenotype has revealed that large deletions removing both Comm and Comm2 act as dominant enhancers of the Netrin mutant phenotype. Further analysis demonstrates that mutations in comm and comm2 individually enhance the Netrin deficiency phenotype, but to a lesser extent than the deletion of both comm and comm2. This result strongly suggests that Comm2 is playing commissural axon guidance at the midline. Uncovering the role of Comm2 will contribute to our knowledge of axon guidance decisions at the midline.
Advisors/Committee Members: Seeger, Mark.
Subjects: Biology; Genetics; Molecular biology
More Like This
25.
Justiniano, Steven E.
IN VITRO SCREEN FOR ROLES OF Drosophila melanogaster TUMOR SUPPRESSORS IN CELL LINE ESTABLISHMENT AND DIFFERENTIAL EXPRESSION ANALYSIS OF Drosophila melanogaster CELL LINES.
Degree: PhD, Molecular Genetics, 2008, Ohio State University
► Research using the fruit fly Drosophila melanogaster has enhanced our understanding of…
(more)
▼ Research using the fruit fly Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and the genetics of early embryonic development. These breakthroughs led to additional discoveries, including the mechanisms of body patterning, organogenesis and control of proliferation. Although great progress has been made using the whole fly as a model for development and disease, in vitro experiments have also contributed to our understanding of biological processes. Cell lines are important tools for biologists primarily because millions of homogeneous cells can be cultured under defined conditions and analyzed. This is particularly important for biochemical analysis. More recently the use of RNAi in cell lines, a mechanism for gene silencing, has allowed the systematic examination of the entire genome in vitro. These RNAi screens have resulted in the discovery of functions for uncharacterized genes. These assays depend critically on the existence of appropriate cell lines and only a few relatively uncharacterized Drosophila cell lines exist. This is in part because the mechanisms that lead to the establishment of cell lines are not defined. Currently, cell lines are established by homogenization of embryos or tissues to produce primary cultures, and subsequently subculturing these cells to produce lines. During this time, cultures presumably acquire mutations, leading to the establishment of cells that can survive indefinitely in vitro. Work done on mammalian cells have revealed that genetic modifications can alter cellular activity in vitro. Activation of telomerase, and genetic alteration of oncogenes and tumor suppressors profoundly alters the behavior of cells in culture and leads these cells to acquire characteristics that allow cells to thrive in vitro. In order to identify genetic elements with similar roles in the establishment of Drosophila cell lines, I have used two approaches. The first approach was an in vitro screen of Drosophila tumor suppressors. Tumor suppressors regulate the proliferation of cells through control of the cell cycle or apoptosis, and the loss of these genes usually results in overgrowth of tissues and the formation of tumors. Homologs of mammalian tumor suppressors have been identified in the fruit fly and the loss of function of these genes often results in increased cell proliferation and/or decreased apoptosis, and consequently the growth of tumors. Because of the importance of tumor suppressors in the regulation of cell cycle and/or control of apoptosis, and because it has been observed that loss of tumor suppressors can cause tumor formation in vivo in flies, I examined the effects of loss of various tumor suppressors in vitro. I hypothesize that the loss of tumor suppressor activity in Drosophila cells in vitro results in increased cell proliferation and may help establish immortal cell cultures. I observed a significant decrease in time to confluency in primary cultures with loss of function tumor suppressor genes. Additionally, I observed a very dramatic decrease in time to confluency in primary cultures with the Pten loss of function mutation. The results of loss of Pten on cells in vitro were examined further through the use of western blotting and differential expression analysis of microarrays on a loss of Pten cell line. Pten cell lines show increased activation of Akt and upregulation of genes involved in metabolism of carbohydrates, lipids and amino acids. The second approach I used was to identify genes involved in the establishment of cell lines, and was designed to take advantage of the wealth of information generated from microarray experiments. Comparison of the transcriptional profiles of various cell types was used to identify differentially regulated genes. We identified 52 genes with differential expression between cell lines and embryos/imaginal discs. Among these 52 genes we identified 18 genes, which are differentially expressed as part of the adaptation to the new tissue culture environment. Of the 34 remaining genes 6 genes were identified as being differentially expressed exclusively in cell lines. Additional experiments are underway to determine the significance of the differential expression of these genes and identify genes underlie the immortalization and/or transformation of cell line cells.
Advisors/Committee Members: Simcox, Amanda.
Subjects: Genetics
Keywords: Drosophila melanogaster; tumor suppressors; cell line; Pten; microarray
More Like This
26.
Kasbek, Christopher.
Regulation of Mps1 Stability Controls Centrosome Duplication and Maintains Genomic Integrity.
Degree: PhD, Molecular Genetics, 2010, Ohio State University
► The main function of centrosomes is the facilitation of bipolar spindle assembly,…
(more)
▼ The main function of centrosomes is the facilitation of bipolar spindle assembly, thus segregating DNA equally to two daughter cells. To assemble a bipolar spindle, a cell must duplicate its single centrosome, inherited from the previous cell cycle, once and only once. Overexpression of Plk4, Sas-6, or Mps1 causes the production of extra centrioles in a single cell cycle, suggesting these proteins must be tightly regulated to maintain proper centrosome control. Mps1, a protein kinase, is regulated by protein degradation that is transiently suppressed by Cdk2 phosphorylation. However, the mechanism of Mps1 degradation and its precise role in centrosome duplication is unknown. In this dissertation, we have tested the hypothesis that misregulating Mps1 can lead to centrosome overproduction using mammalian cell culture as a model system. We have found that phosphorylation of Mps1 at T468 by Cdk2 allows the accumulation of a centrosomal pool of Mps1 that is functional in centrosome duplication. Blocking this phosphorylation, using Cdk2 siRNA or a form of Mps1 that cannot be phosphorylated at T468 (T468A), leads to centrosome-specific proteasome degradation of Mps1 and an inability to execute centrosome duplication. Preventing the degradation of Mps1 at centrosomes, by removing the region around T468, or the expression of a form of Mps1 that mimics Cdk2 phosphorylation (T468D), leads to centrosome overproduction in a single cell cycle, called centrosome reduplication. Furthermore, we have identified one Mps1-binding protein, Antizyme (OAZ), involved in the removal of Mps1 from centrosomes. Decreasing the activity of OAZ leads to centrosome reduplication due to increased levels of Mps1 at centrosomes. OAZ binds to the Mps1 degradation signal (the region surrounding T468) and removes Mps1 from centrosomes downstream of the Cdk2 pathway; T468A can accumulate at centrosomes when OAZ activity is depleted, and T468D is unresponsive to increased OAZ activity. Furthermore, the characterization of OAZ as an Mps1 degradation factor elucidated a role for Mps1 in procentriole assembly; OAZ overexpression leads to a defect in Sas-6 distribution to procentrioles. Identification of additional factors involved in Mps1 degradation may further clarify the role of Mps1 in centriole assembly. We then explored the regulation of Mps1 in tumor-derived cell lines. The ability of these cell lines to undergo centrosome reduplication was correlated with misregulation of Mps1; U2OS and 21NT cells, which naturally undergo centrosome reduplication, fail to degrade Mps1 when Cdk2 is inhibited while other cells, which regulate endogenous Mps1 normally, do not undergo reduplication unless stabilized Mps1 is expressed. Further characterization of U2OS and 21NT cells indicated that the misregulation of Mps1 in tumor-derived cells can occur via multiple mechanisms; U2OS cells express the message lacking the degradation signal, while 21NT cells have a defect in the Mps1 degradation machinery. This study reports a correlation between Mps1 misregulation and centrosome defects in tumor cells. However, analysis of centrosomal Mps1 in tumor samples is required to show a role of Mps1 in tumorigenesis. We believe these studies have supported the hypothesis that the regulation of Mps1 is essential in limiting centrosome duplication to once per cell cycle.
Advisors/Committee Members: Fisk, Harold.
More Like This
27.
Kim, Sunghan.
Characterization of ribosomal S6 protein phosphorylation and possible control of ribosome biogenesis in arabidopsis cell culture.
Degree: PhD, Molecular Genetics, 2004, Ohio State University
► Regulation of translation through control of ribosome biogenesis is the primary means…
(more)
▼ Regulation of translation through control of ribosome biogenesis is the primary means of adjusting the growth and proliferation potential of an organism in response to nutrition and abiotic stresses, which is conserved among all eukaryotes. The phosphorylation status of rpS6, one of the small ribosomal subunit proteins, has been identified as a key parameter of this response in which TOR (target of rapamycin) kinase has been implicated as the master regulator. In order to have a better insight to the translational regulation mediated through ribosomal S6 phosphorylation in plants, the phosphorylation status of rpS6 and its upstream regulator kinase, S6K1, was studied under different physiological conditions, starvation and osmotic stress in particular. A novel kinase assay system which allows pulling down a specific kinase from cells as bound to the specific GST-fusion protein substrate has been developed to facilitate this goal. The results of the kinase assays suggest that the phytohormone auxin and a nutrient sensing signal are acting in parallel to control the pathway regulating rpS6 phosphorylation and translational activity in Arabidopsis cell culture. The same principle of this novel kinase assay method was also applied in a larger scale to identify the kinase as well as any other regulatory elements interacting with the substrate by mass spectrometry. Several Arabidopsis proteins with chromatin related functions were identified as interacting partners of rpS6. Of particular interest among them is a nucleolus targeted histone deacetylase, HD2B, as recent evidences in yeast suggest that TOR controls ribosome biogenesis through modulation of histone deacetlyase activity. Thus, the finding made by this study may provide a clue to explain the molecular mechanism of ribosome biogenesis in which rpS6 plays a crucial role in connecting the functions of TOR and histone deacetylase. Regulation of translation through control of ribosome biogenesis is the primary means of adjusting the growth and proliferation potential of an organism in response to nutrition and abiotic stresses, which is conserved among all eukaryotes. The phosphorylation status of rpS6, one of the small ribosomal subunit proteins, has been identified as a key parameter of this response in which TOR (target of rapamycin) kinase has been implicated as the master regulator. In order to have a better insight to the translational regulation mediated through ribosomal S6 phosphorylation in plants, the phosphorylation status of rpS6 and its upstream regulator kinase, S6K1, was studied under different physiological conditions, starvation and osmotic stress in particular. A novel kinase assay system which allows pulling down a specific kinase from cells as bound to the specific GST-fusion protein substrate has been developed to facilitate this goal. The results of the kinase assays suggest that the phytohormone auxin and a nutrient sensing signal are acting in parallel to control the pathway regulating rpS6 phosphorylation and translational activity in Arabidopsis cell culture. The same principle of this novel kinase assay method was also applied in a larger scale to identify the kinase as well as any other regulatory elements interacting with the substrate by mass spectrometry. Several Arabidopsis proteins with chromatin related functions were identified as interacting partners of rpS6. Of particular interest among them is a nucleolus targeted histone deacetylase, HD2B, as recent evidences in yeast suggest that TOR controls ribosome biogenesis through modulation of histone deacetlyase activity. Thus, the finding made by this study may provide a clue to explain the molecular mechanism of ribosome biogenesis in which rpS6 plays a crucial role in connecting the functions of TOR and histone deacetylase.
Advisors/Committee Members: Verma, Desh.
Subjects: Biology, Molecular
Keywords: ribosomal protein; S6K; TOR; Histone deacetylase
More Like This
28.
Lai, Tsung-Po.
Identification of cis-acting sequences and trans-acting factors for targeting the peripherally associated membrane protein, Trm1-II, to the inner nuclear membrane.
Degree: PhD, Molecular Genetics, 2012, Ohio State University
► Appropriate nuclear membrane structure is important in eukaryotic cells. Significantly, many human…
(more)
▼ Appropriate nuclear membrane structure is important in eukaryotic cells. Significantly, many human diseases are caused by failure to correctly localize inner nuclear membrane (INM) proteins to the nuclear rim. To gain an understanding of the targeting mechanism of peripherally associated INM proteins, I employed the budding yeast Saccharomyces cerevisiae as a model system and Trm1-II, a tRNA modification enzyme that is peripherally bound to the INM, as a reporter. In Saccharomyces cerevisiae, the TRM1 gene codes for the tRNA-specific N2, N2-dimethylguanosine methyltransferase. It is located in mitochondria and nuclei by encoding two isomeric forms of the proteins, Trm1-I and Trm1-II, via alternative translation initiation. The longer form, Trm1-I, is exclusively localized to the mitochondria while the shorter form, Trm1-II, is located in the nucleus (90%) and mitochondria (10%). The nuclear pool of Trm1-II is peripherally associated with the inner nuclear membrane (INM). The mechanism of targeting Trm1-II to the INM is unclear. To characterize the amino acids important for targeting Trm1-II to the INM, I employed random and site-directed mutagenesis, and uncovered specific amino acids necessary for Trm1-II to locate at the INM. I defined a sequence of ~20 amino acids that contains information necessary to target Trm1-II to the INM. To address whether the newly defined region necessary for Trm1-II INM location is also sufficient to target reporter proteins to the INM, I demonstrated that this short peptide causes the redistribution of reporter proteins from the nucleoplasm to the INM. Thus, I identified the first motif for targeting peripherally associated proteins to the INM in yeast (Lai et al., 2009). A genome-wide study that identified subunits of the N-terminal acetyltransferase C (Nat C): Mak3, Mak10, and Mak31 function in Trm1-II INM targeting and suggested N-terminal acetylation of Trm1-II is necessary for it INM location (Murthi and Hopper, 2005). The data suggest that Trm1-II contains information for INM targeting other than the INM targeting motif. To further address the mechanism of Trm1-II INM location, Trm1-II, non-functional INM motif Trm1-II mutant and Trm1-II from cells without Nat C activity were C-terminally tagged with ZZ domain of Protein A (ZZ) or GFP-ZZ to search for Trm1-II interacting partner(s) at the INM using biochemical approaches. Surprisingly, no specific interacting proteins were uncovered by these studies. Rather, the evidence showed that Trm1-II may interact with lipids via the INM targeting motif and associate with the INM. In addition, I identified that without acetylation at N-terminus Trm1-II has the ability to modify tRNAs and to interact with membrane lipids. The data support a model that the INM targeting motif, rather than N-acetylation, is important for Trm1-II INM targeting by facilitating Trm1-II-lipid interaction at the INM. Since non-functional INM targeting motif Trm1-II mutant contains an amino acid substitution, it raise a possibility that the non-functional INM targeting motif Trm1-II mutant may be a misfolded protein, and thereby affecting Trm1-II-lipid interactions. Further investigation are required to confirm that the INM targeting motif is important for Trm1-II-lipid binding and INM targeting.
Advisors/Committee Members: Hopper, Anita.
More Like This
29.
Li, Fu.
THE PTEN-ETS2 SIGNALING AXIS REGULATES MAMMARY TUMORIGENESIS FROM THE STROMAL FIBROBLASTS.
Degree: PhD, Molecular Genetics, 2010, Ohio State University
► Breast cancer remains the second leading cause of cancer related death in…
(more)
▼ Breast cancer remains the second leading cause of cancer related death in women, in spite of advancements in detection, diagnosis and treatment. The heterogeneity of breast cancer hindered our understanding of the mechanisms of this disease. The tumor microenvironment, composed of stromal cells and non-cellular extracellular matrix, represents one of the heterogeneous features of breast cancer. One stromal cell type, fibroblasts, has been proposed to be important in the regulation of the development of normal mammary glands and tumors. However, the exact signaling pathways governing the crosstalk between the fibroblasts and tumor cells remain poorly understood. In the present study, we examined functions of Pten and Ets2 in stromal fibroblasts using conditional knockout mouse models. Deletion of Pten from the stromal fibroblasts resulted in an acceleration of mammary tumorigenesis. Inactivation of Pten in the fibroblasts promoted collagen deposition, inflammation and angiogenesis in the mammary stroma, which in turn supported tumor growth. In addiction, the absence of Pten also resulted in the activation of Ets2, a transcription factor and proto-oncogene, in the stromal fibroblasts. Ets2 acted as a critical downstream effector molecule of Pten, as deletion of Ets2 in fibroblasts reduced tumor growth caused by Pten-absent fibroblasts. Furthermore, Ets2 ablation resulted in a reversal of inflammation and angiogenesis caused by Pten deletion in the fibroblasts. Cre/loxP mediated conditional inactivation of Ets2 in the stromal fibroblasts and mammary epithelial cells revealed that Ets2 was necessary for tumor growth in the fibroblasts, but not the mammary epithelium, in the MMTV-PyMT mouse model. Gene expression profiling revealed that Ets2 controls a tumor fibroblast specific transcriptional program to mediate angiogenesis and ECM remodeling in the tumor microenvironment. As a transcription factor, Ets2 directly controlled the transcription of target genes, such as Mmp9, in the tumor associated fibroblasts. Ets2-null tumor associated fibroblasts had impaired ability to recruit endothelial cells and produce MMP9. Importantly, both the mouse Pten fibroblast gene signature and Ets2 tumor fibroblast signature were able to separate human breast cancer stroma from normal stroma, suggesting the PTEN-ETS2 axis represents a major pathway deregulated in breast cancer stroma. Post-translational regulation of Ets2 by PTEN has been shown to be mediated by AKT, ERK and JNK pathways. However, the transcriptional regulation of Ets2 downstream of PTEN was unknown. With a panel of chemical inhibitors and ShRNA knockdowns, we have found that the activation of the JNK pathway was critical for the upregulation of Ets2 mRNA in Pten-deficient fibroblasts. Additional results suggest JNK is the main functional pathway controlling Ets2 expression in Pten-deficient stromal fibroblasts. In summary, we have identified that PTEN and ETS2 as key regulators in the stromal fibroblasts to modulate mammary epithelial tumor growth. Understanding how the PTEN-ETS2 axis acts in the stroma provided novel mechanisms of cancer epithelium-stroma interactions.
Advisors/Committee Members: Ostrowski, Michael.
Subjects: Biology; Biomedical research
Keywords: Stromal fibroblasts, Pten, Ets2
More Like This
30.
Li, Jing.
Biological function of E2F7 and E2F8 is essential for embryo development.
Degree: PhD, Molecular Genetics, 2009, Ohio State University
► The novel E2F7 and E2F8 family members are thought to function as…
(more)
▼ The novel E2F7 and E2F8 family members are thought to function as transcriptional repressors important for the control of cell proliferation in vitro. However, as the most recently indentified and least studied E2F members, their biological functions in vivo remain unknown. Here we have analyzed the consequences of inactivating E2f7 and E2f8 in mice. While loss of either E2f7 or E2f8 did not significantly affect mouse development, their combined ablation resulted in massive apoptosis, dilated blood vessels and severe placental defects, culminating in embryonic lethality by day 11.5. E2F7 and E2F8 formed homo-dimers and hetero-dimers that could recruit various co-repressor complexes to E2F binding sites of target promoters, including E2f1. Consistent with their important role in transcriptional repression, mouse embryonic fibroblasts (MEFs) deficient for E2f7 and E2f8 expressed abnormally high levels of E2f1 and other E2F-target mRNAs. These double knockout MEFs proliferated surprisingly well, but accumulated high levels of p53 protein and were hypersensitive to DNA damage-induced cell death. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis observed in double mutant embryos but failed to rescue their embryonic lethality.In order to identify the leading cause of fetal death, the critical tissues, related cellular processes and molecular mechanisms of E2F7 and E2F8 function, we utilized conditional knockout strategies to show that extra-embryonic function of E2F7 and E2F8 is both necessary and sufficient for embryo development, and thus define the placental abnormalities as the leading cause of embryonic lethality in E2f7-/-E2f8-/- embryos. Consistent with this genetic finding, cellular examination of double mutant placentas revealed ectopic DNA replication and inappropriate mitosis in certain cell lineages of placenta. We also provided two distinct molecular mechanisms of E2F7 and E2F8 function to explain these cellular phenotypes. On one hand, E2F7 and E2F8 could directly repress a novel transcription network that is critical for controlling DNA replication. On the other hand, E2F7 and E2F8 could indirectly regulate the expression of mitotic cyclins and therefore coordinate mitosis progression. We believe disruption of these pathways in E2f7-/-E2f8-/- placentas culminates in G1-S and G2-M specific defects and, presumably, leads to the profound placental abnormality and its associated fetal death. In summary, this study clearly provides the first in vivo evidence for the biological functions of E2F7 and E2F8. We demonstrate, as a unique repressive arm of the E2F program, E2F7 and E2F8 are not only critical for the control of apoptotic in the fetus, but also essential for the regulation of cell cycle progression in the placenta. We conclude that the synergistic function of E2F7 and E2F8 is essential for embryo development.
Advisors/Committee Members: Leone, Gustavo.
Subjects: Genetics; Molecular biology
Keywords: E2F; embryo development; apoptosis; cell cycle; transcriptional regulation
More Like This
[1] [2] [3]
