Tsonwin Hai - US grants

Long term objective: To better understand the regulation of eukaryotic gene expression. From atherosclerosis to cancer, many diseases are the result of under-expression or over-expression of a gene or a group of genes. Thus, a better understanding of gene expression will lead to a better understanding of, and possibly even cures to, different diseases. Specific aim: To study the molecular mechanisms by which a group of activating transcription factor (ATF) dimers regulate transcription. One recent discovery in transcription research is that many different transcription factors can bind to a given DNA regulatory sequence in vitro. Since a given regulatory sequence may occur in different promoters, this discovery raises several questions. For example, do these different transcription factors regulate different promoters? If so, how is the specificity achieved? Do they differ from one another in ways other than regulating different promoters? This proposal addresses these questions by describing experiments to study a superfamily of transcription factors: the ATF/CREB and Fos/Jun "leucine zipper" proteins. These transcription factors form selective dimers with each other via the leucine zipper regions, and bind to similar DNA sequences. In addition, they are induced by many extracellular stimuli, such as viral infection, growth factors and peptide hormones that increase cellular cAMP level. The goal of this proposal is to use ATF as a model to elucidate fundamental principles of transcriptional regulation. The strategy is to focus on a group of stable ATF homodimers and heterodimers: ATF-1, ATF-3, ATF-4, ATF-3/c-Jun and ATF-4/Fra-1. Specific methods: (1) A "random mutagenesis" analysis to study the importance of DNA sequences on ATF dimer binding, (2) In vitro transcription and in vivo transfection experiments to find out whether different ATF dimers regulate different genes; (3) Pulse-chase labeling, chemical cross-linking, immunoprecipitation and two-dimensional analysis to study how different ATF proteins are induced to become transcriptionally active by extracellular stimuli.

The proper execution of organogenic processes, such as neurogenesis, requires the coordinated activities of various regulatory proteins during development. The pan-neural expressed protein Prospero (Pros) represents a predominately nuclear localized, atypical homeodomain protein, that is required for the proper expression of an array of other regulatory genes during the formation of the nervous system in Drosophila. As a consequence, loss of Prospero activity results in severe defects in the developing nervous system and death of the embryo during the late stages of embryogenesis. Previous work has shown that Prospero can function as a transcription factor, and the capacity of Pros to modulate the activities of several other homeodomain transcription factors. The present proposal is aimed towards the molecular genetic analysis of the structure/functional properties of the pros protein in its in vivo interactions with homeodomain proteins during neurogenesis. High degrees of evolutionary conservation have been observed for regulatory proteins, including Pros, between vertebrate and invertebrate systems. The analysis of the specific functional requirements and interactions in a model system such as Drosophila provides information that is of direct relevance for the understanding of the functional roles of corresponding vertebrate genes in normal development and pathological processes.

Abstract Sack
A Wide-Field Deconvolution Microscope for Cell and Developmental Biology

Deconvolution microscopy provides superior spatial resolution (especially at high magnification) that is ideal for co-localizing multiple fluorescent probes in fixed and living cells in three dimensions. A wide-field deconvolution microscopy facility will be established at Ohio State University. The system will be configured around a Zeiss Axiophot 2 microscope with motorized stage, a high-resolution cooled CCD camera, and integrated software for image acquisition and deconvolution.
Current core users will focus on the cell and developmental biology of plant, neurobiological and fungal material. Specific projects include: (1) the molecular functions of genes controlling cell proliferation, terminal differentiation, and axonal outgrowth in the Drosophila nervous systems, (2) the cell biology and regulation of plant vesicular trafficking and cytokinesis, (3) the functions and localization of g-tubulin in Arabidopsis and Aspergillus, (4) genes and mechanisms controlling stomatal patterning and development in Arabidopsis, (5) the molecular genetic regulation of axonal outgrowth in zebrafish, (6) functional interactions between plant nuclear matrix proteins, and (7) the differential spatial regulation of protein and mRNA localization within mouse Purkinje cells.
Deconvolution imaging is highly likely to advance the understanding of the function and localization of specific proteins in several model systems. This facility will enhance interactions between cell and developmental biologists in the College of Biological Sciences, the Plant Biotechnology Center and the Neurobiotechnology Center. It will also contribute substantively to the training of post-doctoral researchers, graduate students and undergraduate students.

During development of multicellular organisms, regulatory systems governing cell proliferation and developmental programs regulating the formation of specific organs, tissues or cell types, have to interface to ensure that an appropriate number of cells are generated for every tissue and organ. Equally important is the regulated termination of cell proliferation and initiation of lineage appropriate differentiation. A substantial body of work has provided a rather detailed picture of the regulatory systems controlling cell proliferation and cell cycle arrest. In contrast rather little is known, how tissue or lineage specific developmental programs functionally interact with cell cycle regulatory system. In the fruit fly Drosophila melanogaster, the pan-neural expressed neuronal precursor gene prospero (pros) is critical for proper termination of cell proliferation and initiation of neuronal differentiation during embryonic neurogenesis. In this role, pros activity is required for the proper transcriptional regulation of multiple key cell cycle regulatory genes, including the cyclin dependent kinase (cdk) inhibitor gene dacapo, the cdc25 gene string, E2F and cyclin E. Two additional pan-neural neuronal precursor genes, deadpan (dpn) and asense (ase), have been shown to be critical for cell proliferation during larval optic lobe development and proper expression of the cdk inhibitor gene dacapo. This group of pan-neural transcription factor encoding genes represent, or are part of, a critical regulatory link between neuronal lineage specific developmental programs and cell proliferation and/or neuronal differentiation. Experiments conducted under this project will firstly determine, using a range of developmental-genetic approaches, the regulatory capacity and genetic interactions of Pros in the regulation of cell proliferation and differentiation during embryonic and larval neurogenesis. Secondly, functional in vivo and in vitro analysis of transcriptional regulatory regions of dacapo, string and cyclinE will be performed to determine the specific sequence motifs involved in the pros mediated transcriptional regulation of these genes. To this end reporter gene constructs will be used to map and functionally define Pros response elements in transgenic flies. In vitro DNA binding analysis and in vitro mutagenesis experiments will complement the in vivo approaches. These experiments will provide an initial understanding of the functional mode and interactions of this newly emerging regulatory system. As all genes involved in this study are evolutionarily conserved from Drosophila to humans, the information gained from this analysis should have direct relevance for the understanding of similar developmental processes in a number of other organisms.

[unreadable] DESCRIPTION (provided by applicant): Goal and Significance: Diabetes is a major health problem in the U.S. Many years of research indicate that beta cell destruction plays an important role in the pathogenesis and complication of diabetes. The goal of this proposal is to elucidate the roles of Activating Transcription Factor 3 (ATF3), a stress-inducible gene, in stress-induced signal transduction, beta cell dysfunction and destruction. The proposed research will test the following hypotheses. [unreadable] (1) Aim 1: To test the hypothesis that ATF3 is induced in beta cells by stress signals, at least in part, through the MAPK and NFkB pathways. Dominant negative molecules that block the activation of these pathways will be used to determine whether they could inhibit the induction of ATF3 in beta cells by stress signals (IL-1beta, hyperglycemia, and hyperlipidemia). In addition, constitutively active mutants that activate these pathways will be used to determine whether they could induce the expression of ATF3 in the absence of exogenously applied signals. [unreadable] (2) Aim 2: To test the hypothesis that expression of ATF3 leads to beta cell dysfunction, destruction and the development of diabetes. The mifepristone-inducible system will be used to generate transgenic mice expressing ATF3 in the islets in an inducible manner. These mice will be characterized for morphological, immunohistochemical and physiological parameters, and for islet cell death and cell proliferation. [unreadable] (3) Aim 3: To test the hypothesis that ATF3 plays an essential role in cytokine-induced beta cell dysfunction and destruction. Islet cells will be isolated from wild type (ATF3+/+), heterozygous (ATF3+/-) and homozygous (ATF3-/-) ATF3 knockout mice. Cells will be subjected to pro-inflammatory cytokines, nitric oxide donor S-nitroso glutathione (GSNO), or medium (control). Cell death will be analyzed at various time points to determine whether ATF3 is "necessary" for stress signals to induce efficient beta cell death. In addition, islet functions will be analyzed to determine whether ATF3 is necessary for cytokines to induce beta cell dysfunction. For in vivo experiments, wild type and knockout mice will be injected with streptozotocin (STZ) to induce diabetes. Blood glucose levels and diabetes incidence will be analyzed to determine whether ATF3 knockout mice are less sensitive to STZ than wild type mice. [unreadable] [unreadable]

Goal and Hypothesis: The long-term goal of this proposal is to improve the efficiency of islet transplantation. While tremendous progress has been made toward this goal (such as the Edmonton Protocol), two key limitations prevent islet transplantation from being a widespread clinical reality: (1) the need for heavy immunosuppression, and (2) the requirement of large numbers of islets per recipient. This proposal will address these limitations by testing the hypothesis that stress-inducedapoptosis plays an important role in 8 cell destruction during islet transplantation. Twostress-inducible, pro-apoptotic genes will be the focus of the studies: ATF3 and iNOS. Aim 1: To test the hypothesis that the ATF3 does not play a major role in the death receptor- mediated pathway. Caspase 8 is a key molecule to transmit the apoptotic information from the death receptors: Fas and TNFR. Efforts will be made to determine whether ATF3/iNOS-mediated pathway is distinct from death receptor mediated pathway. If they are, inhibition of caspase 8 should further enhance the ability of the ATFS/iNOS knockout islets to resist to stress-induced apoptosis. Aim 2: To test whether islets deficient in ATF3 and/or iNOS have reduced graft rejection. Three experimental islet transplant models will be used to determine whether the lack of ATF3 and/or iNOS alleviate(s) any of the main obstacles for islet graft survival: primary non-function (by syngeneic model), allo-immunity (by allogeneic model) andauto-immunity (by auto-immune model). Aim 3: To test gene silencing in the islets by RNA interference (RNAi) - feasibility test using ATF3 and iNOS as target genes. DMA constructs expressing short hairpin RNAs under the control of the U6 promoter will be generated to target the degradation of ATF3 or/and iNOS mRNA. Their efficiency will be tested in the insulin-producing MIN6 Rcells first then in primary islets. If they work, wild type islets with "knockdown" of ATF3 and/or iNOS by RNAi will be tested to determine whether they survive better than islets without the knockdown of these pro-apoptotic genes. Significance: This proposal combines mechanistic studies of 8 cell death with technological development of gene silencing, with the objective to improve islet transplantation. If successful, the proposed research will enhance not only our understanding of islet destruction but also our ability to engineer islets with improved survivability. This, in turn, will enable islets to be grafted at lower numbers per recipient. In addition, because the islets are less vulnerable, they may tolerate the immune attacks remained under the condition of mild immunosuppression, thus avoiding the deleterious effects of heavy immunosuppression commonly used in transplantation.

[unreadable] DESCRIPTION (provided by applicant): Goal and Hypothesis: The long-term goal of this proposal is to improve the efficiency of islet transplantation. While tremendous progress has been made toward this goal (such as the Edmonton Protocol), two key limitations prevent islet transplantation from being a widespread clinical reality: (1) the need for heavy immunosuppression, and (2) the requirement of large numbers of islets per recipient. This proposal will address these limitations by testing the hypothesis that stress-induced apoptosis plays an important role in 8 cell destruction during islet transplantation. Two stress-inducible, pro-apoptotic genes will be the focus of the studies: ATF3 and iNOS. Aim 1: To test the hypothesis that the ATF3 does not play a major role in the death receptor-mediated pathway. Caspase 8 is a key molecule to transmit the apoptotic information from the death receptors: Fas and TNFR. Efforts will be made to determine whether ATF3/iNOS-mediated pathway is distinct from death receptor mediated pathway. If they are, inhibition of caspase 8 should further enhance the ability of the ATFS/iNOS knockout islets to resist to stress-induced apoptosis. Aim 2: To test whether islets deficient in ATF3 and/or iNOS have reduced graft rejection. Three experimental islet transplant models will be used to determine whether the lack of ATF3 and/or iNOS alleviate(s) any of the main obstacles for islet graft survival: primary non-function (by syngeneic model), allo-immunity (by allogeneic model) and auto-immunity (by auto-immune model). Aim 3: To test gene silencing in the islets by RNA interference (RNAi) - feasibility test using ATF3 and iNOS as target genes. DMA constructs expressing short hairpin RNAs under the control of the U6 promoter will be generated to target the degradation of ATF3 or/and iNOS mRNA. Their efficiency will be tested in the insulin-producing MIN6 R cells first then in primary islets. If they work, wild type islets with "knockdown" of ATF3 and/or iNOS by RNAi will be tested to determine whether they survive better than islets without the knockdown of these pro-apoptotic genes. Significance: This proposal combines mechanistic studies of 8 cell death with technological development of gene silencing, with the objective to improve islet transplantation. If successful, the proposed research will enhance not only our understanding of islet destruction but also our ability to engineer islets with improved survivability. This, in turn, will enable islets to be grafted at lower numbers per recipient. In addition, because the islets are less vulnerable, they may tolerate the immune attacks remained under the condition of mild immunosuppression, thus avoiding the deleterious effects of heavy immunosuppression commonly used in transplantation. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Goal and Hypothesis: During cancer progression, the cells encounter many stress signals. If beyond repair, the cells have built-in mechanisms to eliminate themselves. The successful cancer cells managed to foil this hardwired stress response. In fact, it appears that cancer cells can co-opt some tumor suppressors to become oncogenes. TGF[unreadable] is the best-known example exhibiting this "Jekyll and Hyde" conversion. ATF3, a stress- inducible gene, is a regulatory gene that was recently identified to have a dichotomous role in cancer progression: it is pro-apoptotic in non-transformed breast epithelial cells, but protects the malignant cells from stress and promotes their metastasis. The long-term objective is to understand the cancer dichotomy using ATF3 as a handle to address this issue. This proposal will focus on the oncogenic aspect of ATF3 in breast cancer. Aim 1 will test the hypothesis that the interaction of ATF3 with Smad3, a protein in the TGF[unreadable] pathway, plays an important role in the oncogenic activity of ATF3 in advanced breast cancer cells. This will be tested by structure-function analyses, including domain swap and site-directed mutagenesis. Aim 2 will test the hypothesis that ATF3 exerts its oncogenic action in malignant cells, at least in part, by regulating downstream target genes. Potential target promoters will be tested by chromatin immunoprecipitation assay and transcription assay to determine whether they are the direct target genes of ATF3. In addition, the biological significance of their regulation by ATF3 will be tested. Aim 3 will test the hypothesis that ATF3 is important for macrophage-cancer interaction. Both gain- and loss- of-function approaches, using in vitro co-culture and in vivo fat pad injection models, will be taken to test whether ATF3 plays a role in the ability of cancer cells to interact with macrophages. Significance: ATF3 is a new regulator that has a dichotomous role in cancer progression, and may play a role in stroma-cancer interaction. Because it is induced by anti-cancer drugs, its oncogenic function indicates that these drugs may have undesired effects. Information from the proposal may provide clues for rational designs of anti-cancer treatment, thus potentially changing the clinical practice in the future. PUBLIC HEALTH RELEVANCE: Despite the tremendous advances in early detections and treatments, breast cancer becomes incurable once metastasized beyond the regional lymph nodes. Thus, to combat breast cancer, it is essential to better understand its metastasis. This proposal investigates a master switch gene that regulates breast cancer metastasis.

DESCRIPTION (provided by applicant): The environment to which humans are exposed often contains toxins and stress factors, which induce a variety of biological responses. These are gene-directed active processes; thus, how the cells respond and how genes and environments interact play an important role in disease development. The goal of this R21 is to establish a mouse model to study the biological responses to various environmental stresses. Designs and Utilities: The design is such that it will allow the researchers to trace and study both the cells under stress at the time of harvesting the tissues and the cells that were under stress in their past - long before harvesting. The mice will carry two transgenic alleles. 1. Stress-inducible Cre*: encodes a recombinase Cre fusion (Cre* for short), whose expression is turned on by a broad spectrum of stress signals, and 2. ROSA-Reporter: encodes Yellow Fluorescent Protein (YFP) preceded by Lox-STOP-Lox (LSL). The production of YFP is blocked by LSL and is possible only if the STOP is removed by active Cre*. This event physically and thus permanently alters the DNA, resulting in YFP production (green) to forever mark the cells. This allows one to trace the cells and determine their fate. The Cre* is fluorescent (red); thus, the cells can be labeled red, if they are actively under stres (producing Cre*). With proper designs, one can distinguish cells that were once under stress in their past from those currently under stress. The fluorescent colors allow not only cell imaging but also cell isolation (by sorting) for biochemical and molecular analyses. Aim 1: To generate and characterize the stress-inducible Cre* transgenic mice. The transgenic construct will be generated using BAC recombineering and injected into pronucleus to make the mice. The mice will then be characterized for the expression and half-life of Cre* under acute and chronic stress paradigms. Aim 2: To generate and characterize the double transgenic mice. The stress-inducible Cre* mice will be crossed with the YFP reporter mice (available) to generate the double transgenic mice. The mice will then be analyzed to determine how tight the system is and how efficient the active Cre* is in removing the STOP. Innovation and Significance: The mouse model is novel for its ability to trace the fate of cells under various stresses, such as those induce liver injury, heart attack, neuronal degeneration, and cancer development. In addition, the proposed mouse model allows researchers to address issues regarding the transition from acute to chronic (or repeated) injuries¿an important but not well understood issue. The broad inducibility of Cre* makes the model an excellent tool to study environmentally-induced diseases - a special emphasis of NIEHS.

DESCRIPTION (provided by applicant): Numerous neurological diseases and pathologies include, or are hypothesized to include, cellular injury and/or stress as part of the mechanism. Experimental approaches to examine these mechanisms, or even determine if they are indeed at play in certain conditions, are generally limited. They include post-mortem analyses of known stress-response genes/molecules, in vivo pharmacological manipulations which are often systemic, or functional assessments/manipulations which often cannot separate stressed from non-stressed neurons. Ideally, it would be possible to determine, a priori on a cell-by-cell basis, which neurons in a mixed population were exhibiting a cell-stress response. Further, since some stress responses, or at least some components of the stress response, are only transiently expressed even though the overall stress responses may have a cumulative effect on the cell, it would be highly useful to have an a priori indication of which neurons had exhibited a stress response at some point in the past. It would also be highly useful to be able to selectively assess the cellular and inter-cellular functionality of the stressed neurons. To these ends we propose to generate new functional-reporter transgenic mouse lines which will incorporate these characteristics. The reporters will be driven by the promoter region of Activating Transcription Factor 3 (ATF3), a hub protein involved in a variety of cellular stress responses. Generation of the mice will be via BAC-transgenes in order to preserve function of the native ATF3 alleles, which are necessary for certain cellular processes. The BAC transgene with the ATF3 locus will drive production of the light-gated ion channel channelrhodopsin-2 (ChR2) fused to a fluorescent protein. One model will have the reporters produced in an analogue fashion (i.e., to reflect native ATF3 expression). A second model will use an inducible Cre-recombinase system to permanently switch-on reporter production, thus allowing, at any later time, identification of neurons that previously expressed a stress response. These functional-reporter models will allow offline anatomical assessments, FACS or laser-capture separations, fate-tracing of previously-stressed neurons, and in vivo or in vitro functional assessments specifically of stressed neurons (i.e., those expressing ChR2).