DEVELOPMENTAL RESEARCH PROGRAM
Ramesh A Shivdasani
Dana-farber Cancer Institute, 44 Binney St, Boston, Ma 02115
Abstract: Career development is a particularly important activity for translational research programs. By its very nature, translational research is multidisciplinary. Because of the depth of knowledge required of each individual discipline, our present educational structure seldom allows trainees to function in a multidisciplinary environment. This Career Development Awards Program is designed to provide the incentive and structure necessary to bring junior investigators into the multidisciplinary translational research arena. The key to success of this endeavor is the availability and willingness of mentors throughout DF/HCC to meet the challenges of translational research. This Program will be led by Dr. Monica Bertagnolli, Program Director of the Feinberg Surgical Oncology Fellowship at Brigham and Women´s Hospital and Co- Pi of the DF/HCC SPORE, and Dr. Robert Mayer, Vice-Chair for Academic Affairs at the Dana-Farber Cancer Institute (DFCI) and Director of the DF/PCC Medical Oncology Fellowship. They are joined by a committee including leaders in the fields of cancer therapeutics (Drs. Chabner and Schnipper), Gl cancer surgery (Drs. Warshaw and Zinner), tumor biology and genetics (Drs. Livingston, Kucherlapati, and Podolsky), cancer epidemiology (Dr. Willett), and women´s health and health care disparities (Dr. Bigby). These outstanding senior mentors, representing BWH, MGH, DFCI, and BIDMC, will direct a program that ensure optimal selection, support, and oversight of the Gl SPORE Career Development Awards Program
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5P50CA127003-04_0006 (2010): $226313
Sponsored Links Excellgen http://Excellgen.com
DEVELOPMENTAL RESEARCH PROGRAM
Ramesh A Shivdasani, Associate Professor
Dana-farber Cancer Institute, 44 Binney St, Boston, Ma 02115
Abstract: Career development is a particularly important activity for translational research programs. By its very nature, translational research is multidisciplinary. Because of the depth of knowledge required of each individual discipline, our present educational structure seldom allows trainees to function in a multidisciplinary environment. This Career Development Awards Program is designed to provide the incentive and structure necessary to bring junior investigators into the multidisciplinary translational research arena. The key to success of this endeavor is the availability and willingness of mentors throughout DF/HCC to meet the challenges of translational research. This Program will be led by Dr. Monica Bertagnolli, Program Director of the Feinberg Surgical Oncology Fellowship at Brigham and Women´s Hospital and Co- Pi of the DF/HCC SPORE, and Dr. Robert Mayer, Vice-Chair for Academic Affairs at the Dana-Farber Cancer Institute (DFCI) and Director of the DF/PCC Medical Oncology Fellowship. They are joined by a committee including leaders in the fields of cancer therapeutics (Drs. Chabner and Schnipper), Gl cancer surgery (Drs. Warshaw and Zinner), tumor biology and genetics (Drs. Livingston, Kucherlapati, and Podolsky), cancer epidemiology (Dr. Willett), and women´s health and health care disparities (Dr. Bigby). These outstanding senior mentors, representing BWH, MGH, DFCI, and BIDMC, will direct a program that ensure optimal selection, support, and oversight of the Gl SPORE Career Development Awards Program
Keywords: Cancer of the Gastrointestinal Tract; Cancers; Care, Health; Career Development Awards; Career Development Awards and Programs; Career Development Programs K-Series; DF/HCC; Dana-Farber Cancer Institute; Development; Discipline; Ensure; Environment; Fellowship; Female Health; Gastrointestinal Cancer; Genetic; HOSP; Healthcare; Hospitals; Incentives; Individual; Investigators; K-Awards; K-Series Research Career Programs; Knowledge; Malignant Gastrointestinal Neoplasm; Malignant Neoplasms; Malignant Tumor; Malignant neoplasm of gastrointestinal tract; Medical Oncology; Mentors; Nature; Programs (PT); Programs [Publication Type]; Reproduction spores; Research; Research Career Program; Research Career Programs, K-Series; Research Personnel; Researchers; Spores; Structure; Surgical Oncology; Therapeutic; Translational Research; Translational Research Enterprise; Translational Science; Tumor Biology; Woman; Women`s Health; cancer epidemiology; cancer surgery; career development; design; designing; incentive; inducement; malignancy; meetings; multidisciplinary; neoplasm/cancer; oncologic surgery; programs; success; translation research enterprise; willingness
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: PAR-05-156
5P50CA127003-03_0006 (2009): $224254
Grants awarded to Ramesh A Shivdasani
COMPREHENSIVE PHENOTYPIC COMPARISON OF NORMAL AND CANCER STEM CELLS
Ramesh A Shivdasani
Harvard University, 1350 Massachusetts Ave, Cambridge, Ma 02138
Grant 5RC2CA148222-02 from National Cancer Institute
Abstract: Relapse after therapy remains a critical problem for most types of cancer in humans, even when cancer- specific therapy is initially effective. Understanding the basis for cancer resistance and recurrence is a critical pre-requisite for developing more effective treatments. Recent data indicate that in some cancers, including leukemia, colon cancer, and certain brain tumors, a minority of cells in the initial tumor possesses unique properties reminiscent of normal tissue stem cells a capacity for extensive self- renewal and multilineage differentiation potential. Relative drug resistance in these cells may be especially relevant for cancer recurrence. Thus, the precise relationship between these tumor-initiating cells and normal tissue-specific stem or progenitor cells is extremely important to understand in depth, as key differences might be exploited for therapeutic benefit. In this proposal we outline an approach to identify critical genome-wide similarities and differences between normal and cancer stem cells isolated from normal intestine/colorectal adenocarcinoma, neural stem cells/glioblastoma multiforme (GBM) and hematopoietic stem cells/acute myelogenous leukemia (AML). Using well-validated reagents and experimental approaches, we will isolate highly purified and functionally characterized cancer stem cells from primary human tumors and well-defined mouse models of cancer. We will interrogate gene expression, histone and DNA methylation, and microRNA expression for comparison to normal tissue stem cells, which we will isolate in parallel for these studies. Detailed comparative data will be deposited in a well-annotated central database and analyzed in a manner that allows comparison of normal and malignant stem cells from multiple different tissues. The collective data will provide an unprecedented and comprehensive picture of normal and cancer stem cells, highlighting key differences that may be exploited therapeutically. The dataset and concomitant analysis, served on a powerful computational platform and web interface, will serve as an invaluable resource for the stem cell and cancer research communities. To complement these efforts, we will also develop sophisticated mouse reporter models of four signaling pathways that play seminal roles in cancer stem cell biology Wnt/ss-catenin, Sonic hedgehog, Notch, and PIK3CA/FoxO. As our genomic approaches identify new stem cell pathways, in year 2 we will be positioned to develop up to 3 additional mouse reporter models that will both validate our genomic findings and provide critical resources for future study of these and other tumor types. In sum, the studies proposed in this highly collaborative project will provide vital information to aid in developing therapies that might target cancer stem cells while sparing their normal counterparts. The nature, scope and future scientific and economic impact of this work are ideally suited to the goals of the "Grand Opportunities" funding mechanism, and indeed, in-depth comparison of normal and tumor stem cells can only be accomplished under the aegis of such a program. Many cancers including brain tumors, colon cancer and leukemia contain a population of cancer stem cells that are similar to normal stem cells in some respects, including an inherent ability to propagate the tumor; these "cancer stem cells" are likely resistant to current therapies. This proposal will identify the common and shared feature of cancer stem cells and normal stem cells derived from 3 tissues blood, colon and brain. The results of these studies will inform development of new treatment strategies that target cancer but spare normal stem cells, leading to the development of more effective and less toxic cancer treatments
Keywords: AML - Acute Myeloid Leukemia; Astrocytoma, Grade IV; Behavior; Blood; Blood (Leukemia); Blood Precursor Cell; Body Tissues; Brain; Brain Neoplasia; Brain Neoplasms; Brain Tumors; Cancer Treatment; Cancer stem cell; Cancers; Cells; Colon; Colon Cancer; Colon Carcinoma; Colonic Carcinoma; Colony-Forming Units, Neoplastic; Colorectal Adenocarcinoma; Communities; Complement; Complement Proteins; DNA Methylation; Data; Data Banks; Data Bases; Data Set; Databank, Electronic; Databanks; Database, Electronic; Databases; Dataset; Deposit; Deposition; Development; Drug resistance; Encephalon; Encephalons; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Erinaceidae; Exons; Funding; Funding Mechanisms; Future; Gene Expression; Genetic Models; Genomics; Glioblastoma; Goals; Grade IV Astrocytic Neoplasm; Grade IV Astrocytic Tumor; Hedgehogs; Hematopoietic stem cells; Histones; Human; Human, General; Intestinal; Intestines; Investigators; Large Bowel Adenocarcinoma; Large Intestine Adenocarcinoma; Leukemia, Myelocytic, Acute; Leukemias, General; Malignant; Malignant - descriptor; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Mice; Micro RNA; MicroRNAs; Minority; Modeling; Mother Cells; Murine; Mus; Myeloblastic Leukemia, Acute; Myelogenous Leukemia, Acute; Nature; Nervous System, Brain; Neural Stem Cell; Normal Tissue; Normal tissue morphology; Organ; Pathway interactions; Play; Population; Position; Positioning Attribute; Progenitor Cells; Progenitor Cells, Hematopoietic; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Reagent; Recurrence; Recurrent; Relapse; Relative; Relative (related person); Reporter; Research Personnel; Research Resources; Researchers; Resistance; Resources; Reticuloendothelial System, Blood; Role; Seminal; Signal Pathway; Site; Stem Cells, Neoplastic; Stem cells; Sum; Therapeutic; Tissue Sample; Tissues; Tumor Stem Cells; Work; ing; acute granulocytic leukemia; acute myeloid leukemia; acute nonlymphocytic leukemia; anticancer research; anticancer therapy; base; bowel; cancer recurrence; cancer research; cancer therapy; cancer type; clinical data repository; clinical data warehouse; comparative; computational tools; computerized tools; data repository; drug resistant; economic impact; effective therapy; genome-wide; glioblastoma multiforme; histone modification; human tissue; intervention development; leukemia; malignancy; man; man`s; miRNA; mouse model; neoplasm/cancer; nerve stem cell; neural progenitor cells; neuronal progenitor; neuronal progenitor cells; notch; notch protein; notch receptors; novel; pathway; programs; relational database; resistance to Drug; resistant; resistant to Drug; self-renewal; social role; spongioblastoma multiforme; stem; stem cell biology; therapy development; tool; treatment development; treatment strategy; tumor; tumors in the brain; web interface
Relevance: Narrative: Many cancers including brain tumors, colon cancer and leukemia contain a population of cancer stem cells that are similar to normal stem cells in some respects, including an inherent ability to propagate the tumor; these "cancer stem cells" are likely resistant to current therapies. This proposal will identify the common and shared feature of cancer stem cells and normal stem cells derived from 3 tissues: blood, colon and brain. The results of these studies will inform development of new treatment strategies that target cancer but spare normal stem cells, leading to the development of more effective and less toxic cancer treatments
Project start date: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-OD-09-004
5RC2CA148222-02 (2010): $1702016
1RC2CA148222-01 (2009): $1723218
NF E2 AND GLOBIN GENE EXPRESSION AND HEMATOPOIESIS
Ramesh A Shivdasani, Associate Professor Of Medicine
Dana-farber Cancer Institute 44 Binney St Boston, Ma 02115
Grant 5K08HL003290-03 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Globin gene expression in developing erythrocytes is under the control of locus control regions (LCRs), which appear to interact directly with a remarkably limited array of trans-acting factors. One of the most important of these is NF-E2, which is an obligate heterodimer of hematopoietic-specific p45 and ubiquitous p18 subunits. NF-E2 is believed to provide the major enhancer function for transcription of the globin genes. By a strategy involving homologous recombination in embryonal stem (ES) cells we have generated mice lacking p45 NF-E2 and found them to manifest anemia and severe thrombocytopenia in the face of otherwise normal development. The anemia is evident only after birth and the mice usually die of hemorrhage; megakaryocytes are present in slightly increased numbers. These findings have important implications for LCR function and platelet development. This proposal describes studies on these aspects of blood cells in mice lacking p45 NF-E2. We seek to establish the nature and causes of anemia in these mice, to identify cellular compensatory mechanisms for the absence of the factor, to develop a system for studying the erythroid effect without the confounding feature of blood loss, to begin a rational approach toward the molecular study of NF-E2 function in the differentiation of megakaryocytes and other hematopoietic cells, and to examine the effects of the absence of p18 NF-E2 in vivo. Molecular, histologic and ultrastructural studies will be combined with mouse breeding experiments and transgenesis to achieve these aims.
Keywords: anemia, gene expression, globin, hematopoiesis, antiantibody, intrinsic factor, megakaryocyte, molecular pathology, platelet, transcription factor, Mus musculus, laboratory mouse, southern blotting, transgenic animal
Project start date: 1995-05-01
Project end date: 1998-04-30
5K08HL003290-03 (1997): $82836
Gene Expression In The Mammalian Intestine
Ramesh A Shivdasani, Associate Professor Of Medicine
Dana-farber Cancer Institute 44 Binney St Boston, Ma 02115
Grant 5R01DK061139-04 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: Common disorders of the human gastrointestinal (GI) tract, including congenital malformations, inflammatory bowel, celiac and peptic ulcer disease, intestine-specific infections and cancer, result in considerable morbidity and mortality in the United States. Although the pathophysiology of many of these disorders is likely related in fundamental ways to mechanisms of normal gut development, the molecular regulation of cell differentiation and fetal development of the mammalian intestine is poorly understood. This is particularly true for the molecular basis of the interaction between gut epithelium and its underlying mesenchyme, a key regulator of intestinal development and homeostasis. This proposal seeks to exploit recent technological advances to initiate a systematic molecular analysis of mammalian intestinal development and organogenesis. We will begin by examining quantitative and qualitative changes in the gene transcription profile during the critical interval between 13 and 15 days in mouse gestation, when the gut endoderm undergoes an important cellular transition from a poorly differentiated, pseudostratified tissue into a primitive villous epithelium. Preliminary analysis of two separate Serial Analysis of Gene Expression (SAGE) libraries prepared from these developmental stages reveals statistically significant changes in the expression levels of a surprisingly small number of genes, including several putative regulators of intercellular communication, gene transcription, and intracellular signaling. Some of these changes are presumed to drive the acquisition of cellular fate and morphology and tissue form and function in the developing small bowel. The Specific Aims of the project involve more detailed characterization of these SAGE libraries, a unique resource in the field, with a special view toward identifying some of the critical regulators of early intestinal epithelial differentiation and mesenchymal-epithelial interactions. We further propose to examine the sites of mRNA expression for genes predicted to function in intercellular interactions and hence begin to map key candidate signaling pathways in gut development as a prelude to developing practical strategies to examine gene function more directly. Finally, we will expand this analysis in space and in time, by investigating the dynamic alterations in gene expression at two additional stages in small bowel development, and at parallel stages in development of the stomach and colon. The resulting comprehensive analysis of mammalian gut development, quite possibly the first of its kind, will elucidate some essential aspects of normal developmental processes and should contribute to improved understanding and treatment of common disorders of the GI tract.
Keywords: developmental genetics, functional /structural genomics, gastrointestinal system, gene expression, genetic model, genetic regulation, histogenesis, model design /development, cell cell interaction, cytokine, gastrointestinal epithelium, gastrointestinal neoplasm, genetically modified animal, mammalian embryology, mesenchyme, molecular biology information system, protein structure function, transcription factor, tumor progression, embryo /fetus, gene targeting, in situ hybridization, laboratory mouse, neoplastic cell, northern blotting, serial analysis of gene expression
Project start date: 2002-02-01
Project end date: 2008-01-31
5R01DK061139-04 (2005): $299250
5R01DK061139-03 (2004): $299250
5R01DK061139-02 (2003): $292416
Cellular And Molecular Regulation Of Thrombopoiesis
Ramesh A Shivdasani, Associate Professor Of Medicine
Dana-farber Cancer Institute 44 Binney St Boston, Ma 02115
Grant 5R01HL063143-09 from National Heart, Lung, And Blood Institute IRG: HT
Abstract: Blood platelets prevent hemorrhage, mediate diverse pathologic processes, including inflammation and thrombosis, and are frequently reduced in toxic response to cancer therapy. The central role of platelets in common disorders makes it important to know how megakaryocytes (MKs) terminate differentiation and release platelets into the circulation. Microtubule-based MK cytoplasmic extensions known as proplatelets serve as the immediate precursors of blood platelets and their elaboration requires the activities of transcription factors GATA1 and NF-E2. However, most underlying structural and signaling pathways and relevant targets of the critical transcriptional regulators remain unknown. The platelet-specific beta-tubulin isoform beta1 is a major component of proplatelet and platelet marginal microtubules and requires NF-E2 for expression. Beta1 tubulin-/- mice produce few and spherocytic platelets and thus attest to the importance of this lineage-restricted cytoskeletal factor in thrombopoiesis. We have isolated several MK-expressed proteins that interact with the divergent C-terminus of beta1 tubulin and their initial characterization leads to specific hypotheses about their functions in platelet synthesis or function. Additionally, in an effort to generate a cogent molecular classification of individual stages in MK maturation, we have captured gene expression profiles from MK subpopulations isolated using flow cytometry. The preliminary results again suggest roles for selected signaling pathways that may be tested in primary mouse MKs. The Specific Aims of this proposal are (1) To characterize the associations and functions of beta1 tubulin-interacting MK proteins, especially a novel Ran-binding factor RanBPM-2 and the secretory leukocyte protease inhibitor SLPI. (2) To identify the major molecular events corresponding to recognized morphologic and functional transitions in MK maturation and the molecular pathways that are active at different stages in thrombopoiesis. (3) To test the functions of a limited number of genes identified through Aims 1 and 2 in aspects of platelet biogenesis. We are using an effective and flexible strategy to over express genes in cultured primary MKs, followed by functional and structural examination of the consequences. To facilitate the Aims of this project, we also propose to adapt the method to abrogate expression of endogenous genes. We expect the collective results of our studies to advance current understanding of how MKs produce blood platelets.
Keywords: cell differentiation, developmental genetics, genetic regulation, megakaryocyte, molecular biology, platelet, thrombopoiesis, tubulin, biological signal transduction, cell morphology, gene expression, nuclear protein, protease inhibitor, protein isoform, protein localization, protein protein interaction, protein structure function, recombinant protein, RNA interference, confocal scanning microscopy, genetically modified animal, immunofluorescence technique, laboratory mouse, microarray technology, tissue /cell culture, transfection /expression vector
Project start date: 1999-07-01
Project end date: 2008-06-30
5R01HL063143-09 (2007): $364813
5R01HL063143-08 (2006): $375709
5R01HL063143-07 (2005): $384750
2R01HL063143-06 (2004): $378360
Sponsored Links Excellgen http://Excellgen.com
NFE2 AND BETA 1 TUBULIN IN PLATELET DIFFERENTIATION
Ramesh A Shivdasani, Associate Professor Of Medicine
Dana-farber Cancer Institute 44 Binney St Boston, Ma 02115
Grant 5R01HL063143-05 from National Heart, Lung, And Blood Institute IRG: HEM
Abstract: Blood platelets mediate hemostasis as well as diverse pathologic processes, including thrombosis and inflammation, and thrombocytopenia is a frequent, dose-limiting complication of cancer chemotherapy. The importance of platelets in diseases that are common in the United States is thus well recognized. This makes it necessary to understand how terminally differentiated megakaryocytes (MKs) fragment and release blood platelets. Despite the discovery of thrombopoietin (Tpo) and its signal transduction pathways in MKs, the cellular and molecular basis of terminal MK maturation and platelet release remain unknown. Mice lacking the erythro-megakaryocytic transcription factor NF-E2 have severe, usually lethal, thrombocytopenia resulting from a late arrest in MK cytoplasmic differentiation. Hence, they constitute a powerful and unique model to study thrombocytopoiesis, and are an important tool in the P.I. s long-term goal of defining molecular aspects of platelet production. MKs deficient in NF-E2 have a cell-autonomous defect and fail to produce proplatelets, the precursors of blood platelets, in culture. However, the transcriptional targets of NF-E2 and the relevant biochemical pathways of terminal MK differentiation are not known. Initial efforts to identify genes that fail to be expressed in the absence of NF-E2 have led to a MK-and platelet-specific beta tubulin isoform (beta1) that normally appears late in MK differentiation, localizes to proplatelets and platelet marginal bands, and possibly mediates the cytoplasmic reorganization that accompanies platelet release. Beta1 tubulin expression is completely lost in the absence of NF-E2 function. Preliminary data thus strongly suggest that loss of beta1 tubulin expression in MKs might explain in part why blood platelets are not produced in the absence of NF-E2. The Specific Aims of this proposal include determining whether the absence of beta1 tubulin in NF-E2-deficient cells reflects direct regulation by this transcription factor, and whether restoring beta1 tubulin expression in the defective MKs is sufficient to rescue some or all aspects of thrombocytopoiesis. Further, we propose to use gene targeting in mice to test the essential roles of beta1 tubulin in vivo, independent of its putative relation to NF-E2. Finally, we propose to use a previously successful mRNA subtraction strategy to identify and characterize additional molecular targets of NF-E2 that may participate in platelet biogenesis. The results of these studies should substantially improve the current understanding of how MKs produce blood platelets.
Keywords: cell differentiation, platelet, protein structure function, transcription factor, tubulin, gene expression, genetic regulation, leukopoiesis, megakaryocyte, messenger RNA, protein isoform, thrombopoiesis, gene targeting, laboratory mouse, subtraction hybridization, tissue /cell culture
Project start date: 1999-07-01
Project end date: 2004-06-30
5R01HL063143-05 (2003): $325477
5R01HL063143-03 (2001): $308196
5R01HL063143-02 (2000): $299220
1R01HL063143-01 (1999): $290505
CELLULAR AND MOLECULAR REGULATION OF THROMBOPOIESIS
Ramesh A Shivdasani, Associate Professor
Dana-farber Cancer Institute, 44 Binney St, Boston, Ma 02115
Grant 2R56HL063143-10 from Office Of The Director, National Institutes Of Health
Abstract: Blood platelets are required for hemostasis and mediate diverse pathologic processes, including thrombosis and inflammation. Although the origin of platelets in megakaryocytes (MKs) was recognized over a century ago, cellular and molecular mechanisms of platelet assembly and release remain largely obscure. Current understanding and experimental approaches build on a combination of cell biological studies, genetic analysis in mice, and appreciation of congenital human thrombocytopenias such as the May-Hegglin anomaly and other Myh9-related disorders. At the conclusion of an elaborate maturation process, MKs undergo dramatic morphogenesis to extend long, branched structures called proplatelets. Blood platelets are assembled de novo within these cytoplasmic extensions and ultimately released into the circulation. As a result of NHLBI funding through an R01 award that is now in its 9th year, our group has played a prominent part in investigating the mechanisms of this remarkable process, which depends on activity of the transcription factor NF-E2. We seek to renew funding to continue with studies that will significantly advance understanding of how MKs produce and release platelets. First, we recently reported on the surprising result that the Myh9 gene product, non-muscle myosin heavy chain IIA, regulates proplatelet formation negatively and seems to receive signals through the small-GTPase Rho. Our preliminary studies suggest 2 specific hypotheses (1) that myosin-IIA deficiency promotes precocious platelet assembly within immature MKs, and (2) that myosin-IIA inhibition of platelet release is normally lifted when mature MKs encounter the chemokine Sdf-1/CXCL12, which down-regulates MK Rho activity. In Specific Aim 1 we propose to test these hypotheses critically, using multi-photon intravital microscopy and biochemical and functional analysis of signal transduction in cultured primary mouse MKs. Second, although NF-E2 is a seminal transcriptional regulator of late MK maturation and platelet biogenesis, a satisfactory understanding of its mechanisms and transcriptional targets has been elusive. We have combined chromatin immunoprecipitation with hybridization to tiled genome arrays (ChIP-chip methodology) to begin to identify the nearly complete complement of genes that NF-E2 may regulate in MKs. In Specific Aim 2 we propose to solidify and extend our preliminary ChIP-chip findings to characterize the NF-E2 ¿cistrome¿ and the breadth of cellular processes recruited in thrombopoiesis. Our studies have already started to generate specific and powerful hypotheses about particular NF-E2- binding cis-elements in MK-expressed genes. In Specific Aim 3 we will use cell-based reporter assays and locus-targeted mice to confirm the requirements for particular NF-E2 cis-elements in MK gene regulation. The sum of these studies should lead to new insights into physiologic and molecular control of thrombopoiesis
Keywords: Abbreviations; Affect; Area; Assay; Award; Band Shift Mobility Assay; Bandshift Mobility Assay; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biogenesis; Biologic Assays; Biological; Biological Assay; Bizzozero`s corpuscle/cell; Blood Circulation; Blood Platelets; Blood megakaryocyte; Bloodstream; CHIP assay; Cell Communication and Signaling; Cell Function; Cell Process; Cell Signaling; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; ChIP (chromatin immunoprecipitation); Circulation; Complement; Complement Proteins; Cytokines, Chemotactic; Data; Deetjeen`s body; Disease; Disorder; EMSA; Electrophoretic Mobility Shift Assay; Elements; Funding; Gene Action Regulation; Gene Expression; Gene Expression Regulation; Gene Regulation; Gene Regulation Process; Genes; Genetic; Genetic analyses; Genome; Grant; Hayem`s elementary corpuscle; Hemostasis; Hemostatic function; Hereditary; Homologous Chemotactic Cytokines; Human; Human, General; INFLM; IVM; In element; Indium; Inflammation; Inherited; Intercrines; Intracellular Communication and Signaling; Knowledge; Laboratories; Lead; Learning; Life Cycle; Life Cycle Stages; Lifting; MGDF; MGDF Factor; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow platelet; Mediating; Megakaryocyte Colony Stimulating Factor; Megakaryocyte Growth and Development Factor; Megakaryocytes; Megalokaryocyte; Membrane; Method LOINC Axis 6; Methodology; Methods; Mice; Micro-tubule; Microtubules; Mobility Shift Assay; Molecular; Molecular Interaction; Monomeric G-Proteins; Monomeric GTP-Binding Proteins; Morphogenesis; Murine; Mus; Muscle; Muscle Tissue; Mutate; Myeloproliferative Leukemia Virus Oncogene Ligand; Myosin A; Myosin Heavy Chains; Myosin IIA; Myosin Type IIA, Non-Muscle; NF-E2 protein; NF-E2 transcription factor; NFE2 protein; Nonmuscle Myosin Type IIA; Origin of Life; Output; Pathologic Processes; Pathological Processes; Pb element; Physiologic; Physiological; Platelets; Play; Process; Proteins; Recruitment Activity; Regulation; Reporter; Reporting; Reticuloendothelial System, Platelets; Role; SIS cytokines; Seminal; Signal Transduction; Signal Transduction Systems; Signaling; Small G-Proteins; Small GTPases; Staging; Standards; Standards of Weights and Measures; Structure; Subcellular Process; Sum; System; System, LOINC Axis 4; THPO; Testing; Thrombocytes; Thrombocytopoiesis; Thrombocytopoiesis-Stimulating Factor; Thrombocytopoietin; Thrombopoiesis; Thrombopoietin; Thrombosis; Time; Transfection; base; biological signal transduction; c-mpl Ligand; cell type; chemoattractant cytokine; chemokine; chromatin immunoprecipitation; concept; disease/disorder; gel shift assay; gene product; genetic analysis; genome, mouse; heavy metal Pb; heavy metal lead; insight; interest; intravital microscopy; life course; membrane structure; mouse genome; mpl Ligand; multi-photon; myosin heavy chain; nuclear factor-erythroid 2; recruit; response; rho; shRNA; short hairpin RNA; small hairpin RNA; social role; thrombocyte/platelet; transcription factor
Project start date: 1999-07-01
Project end date: 2010-06-30
Budget start date: 1-JUL-2008
Budget end date: 30-JUN-2010
PFA/PA: PA-07-070
2R56HL063143-10 (2008): $0
5R01HL063143-12 (2010): $426517
2R01HL063143-11A1 (2009): $422947
ANALYSIS OF INTESTINAL GENES REGULATED BY THE TRANSCRIPTION FACTOR CDX2
Ramesh A Shivdasani
Dana-farber Cancer Institute, 44 Binney St, Boston, Ma 02115
Grant 1R01DK082889-01A1 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Appreciation of the molecular mechanisms responsible for intestine-specific gene regulation and cell differentiation is limited. One important regulator, the transcription factor CDX2, is restricted to intestinal epithelium, where it is expressed throughout the crypt-villus unit and required in vivo for differentiation of gut- specific columnar epithelium. Knowledge of CDX2´s transcriptional targets and mechanisms is incomplete, as is understanding of how it functions as a master transcriptional regulator. We have used whole-genome chromatin immunoprecipitation (ChIP) to identify, with high confidence, regions of CDX2 occupancy in colonic epithelial cells. CDX2-binding regions are highly conserved and show significant clustering of motifs for a handful of other sequence-specific DNA-binding proteins previously implicated in intestinal gene regulation. Our studies hence correctly identify numerous CDX2 targets and reveal 3 specific candidate partner transcription factors in intestine-specific gene regulation. We will extend the preliminary data and insights to test specific hypotheses on CDX2 function and molecular mechanisms. Aim 1 seeks to identify which among ~1,100 CDX2-binding sites are bona fide cis-elements in intestine cells. We will identify transcripts that respond to CDX2 depletion in cells that express the factor and to forced CDX2 expression in cells that don´t. We will also test putative cis-elements in functional reporter assays and critically examine whether CDX2 target genes reflect the activities expected of a master regulator. Unexpectedly, we find that CDX2 commonly occupies DNA very close to binding sites for Tcf proteins, transcriptional effectors of the canonical Wnt pathway. Wnt signals are transmitted in many tissues but are critical in intestinal homeostasis. We will test the novel hypothesis that CDX2 imparts intestinal specificity within a global Wnt response. We also find significant co-occupancy of the nuclear receptor HNF4) near CDX2-activated genes and of GATA proteins near genes that CDX2 appears to repress. Aim 2 takes several approaches to test the hypothesis that HNF4) and GATA factors combine with CDX2 to activate and silence genes, respectively. Lastly, genome-wide ChIP on isolated mouse intestinal crypt and villus fractions implies that Cdx2 controls distinct genes within these two functional compartments. In Aim 3 we will test this hypothesis and address the underlying mechanisms. We will delineate Cdx2 partner proteins and ask if Cdx2 binding is needed to generate crypt- and villus-specific chromatin domains or, conversely, if Cdx2 responds to the creation of such domains by other factors. To this end, we have established the feasibility of whole-genome analysis of informative chromatin marks and generated mice in which intestinal Cdx2 levels can be modulated. These studies represent a detailed and comprehensive approach to elucidate mechanisms of intestine-specific gene regulation. Common disorders of the gastrointestinal (GI) tract, including inflammation, malabsorption, ulcers and cancer, cause considerable suffering and death. Although these conditions are fundamentally related to mechanisms of normal gene expression and cell behavior, there is limited appreciation of their molecular underpinnings. The CDX2 protein, which is found almost exclusively in the intestine, is a pivotal regulator in normal and disease states. Investigation of its regulatory functions will improve understanding and, ultimately, treatment of many common GI diseases
Keywords: Abbreviations; Absorption; Address; Alimentary Canal; Anaplastic; Assay; Autoregulation; Binding; Binding (Molecular Function); Binding Sites; Bioassay; Biologic Assays; Biological Assay; Body Tissues; CDX-3; CDX-3 Gene; CDX2; CDX2 gene; CDX2 protein; CDX3; CDX3 Gene; CHIP assay; Caco-2 Cells; Cancer Cause; Cancer Etiology; Caudal Type Homeobox Transcription Factor 2; Caudal-Type Homeo Box Transcription Factor 2; Caudal-Type Homeo Box Transcription Factor 2 Gene; Caudal-Type Homeo Box Transcription Factor 3; Caudal-Type Homeo Box Transcription Factor 3 Gene; Caudal-Type Homeobox Protein 2; Caudal-Type Homeobox Protein 2 Gene; Cdx-2 protein; Cell Communication and Signaling; Cell Differentiation; Cell Differentiation process; Cell Line; Cell Lines, Strains; Cell Signaling; CellLine; Cells; Cessation of life; ChIP (chromatin immunoprecipitation); Chromatin; Closure by Ligation; Columnar Epithelium; Combining Site; DNA; Data; Data Banks; Data Bases; Data Set; Databank, Electronic; Databanks; Database, Electronic; Databases; Dataset; Death; Defect; Deoxyribonucleic Acid; Differential Gene Expression; Digestion; Digestive Tract; Disease; Disorder; Elements; Epithelial; Epithelial Cells; Epithelium, Intestinal; Family; GI Tract; GWAS; Gastrointestinal Diseases; Gastrointestinal Diseases and Manifestations; Gastrointestinal Tract; Gastrointestinal tract structure; Gene Action Regulation; Gene Expression; Gene Expression Regulation; Gene Inactivation; Gene Products, RNA; Gene Regulation; Gene Regulation Process; Gene Silencing; Gene Targeting; Genes; Genetics, in situ Hybridization; Genome; Genomics; Gut Epithelium; Heart; Homeo Boxes; Homeobox; Homeobox Protein CDX-2; Homeobox Protein CDX-2 Gene; Homeostasis; Human; Human Genome; Human, General; INFLM; Immune Precipitation; Immunoprecipitation; In Situ Hybridization; In element; Indium; Inflammation; Insulin-Regulating Transcription Factor CDX3; Insulin-Regulating Transcription Factor CDX3 Gene; Intermediary Metabolism; Intestinal; Intestines; Intracellular Communication and Signaling; Investigation; Knowledge; Life; Ligation; METBL; Malabsorption Syndromes; Mammals, Mice; Man (Taxonomy); Man, Modern; Maps; Measures; Mediating; Metabolic Processes; Metabolism; Mice; Modeling; Molecular; Molecular Interaction; Murine; Mus; Nuclear Receptors; Pathway interactions; Physiologic; Physiological; Physiological Homeostasis; Process of absorption; Proteins; RNA; RNA, Non-Polyadenylated; Reactive Site; Regulation; Regulatory Element; RegulatoryElement; Reporter; Research Proposals; Resolution; Ribonucleic Acid; Role; Sequence-Specific DNA Binding Protein; Signal Transduction; Signal Transduction Systems; Signaling; Site; Specificity; Structure of intestinal epithelium; System; System, LOINC Axis 4; TCF-4; TCF4; TCF7L2; TCF7L2 gene; Targetings, Gene; Testing; Tissue-Specific Differential Gene Expression; Tissue-Specific Gene Expression; Tissues; Transcript; ULCN; Ulcer; Ulceration; Undifferentiated; Villus; absorption; alimentary tract; base; biological signal transduction; bowel; cdx-2 gene product; cell behavior; chromatin immunoprecipitation; chromatin modification; clinical data repository; clinical data warehouse; combinatorial; computational tools; computerized tools; cultured cell line; data repository; digestive canal; disease/disorder; gastrointestinal absorption disorder; gastrointestinal disorder; gastrointestinal epithelium; gene product; genome wide association scan; genome wide association studies; genome wide association study; genome-wide; genome-wide analysis; genome-wide scan; genomewide association scan; genomewide association studies; genomewide association study; genomewide scan; improved; in situ Hybridization Staining Method; in vivo; insight; intestinal crypt; intestinal epithelium; intestinal malabsorption; malabsorption; novel; pathway; public health relevance; relational database; response; social role; transcription factor; transcription factor CDX2; whole genome association studies; whole genome association study
Relevance: Common disorders of the gastrointestinal (GI) tract, including inflammation, malabsorption, ulcers and cancer, cause considerable suffering and death. Although these conditions are fundamentally related to mechanisms of normal gene expression and cell behavior, there is limited appreciation of their molecular underpinnings. The CDX2 protein, which is found almost exclusively in the intestine, is a pivotal regulator in normal and disease states. Investigation of its regulatory functions will improve understanding and, ultimately, treatment of many common GI diseases
Project start date: 2010-09-01
Project end date: 2015-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
1R01DK082889-01A1 (2010): $426232
HEDGEHOG SIGNALING IN EARLY DEVELOPMENT OF THE GASTROINTESTINAL TRACT
Ramesh A Shivdasani
Dana-farber Cancer Institute, 44 Binney St, Boston, Ma 02115
Grant 5R01DK081113-02 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Although many gastrointestinal (GI) disorders are intimately related to cellular relationships established during embryogenesis, current understanding of GI developmental mechanisms is incomplete. GI tract organogenesis requires exchange of signals between cells derived from gut-specific endoderm and the adjacent splanchnic mesoderm; investigating the nature and mechanisms of this exchange is important. Among the handful of pathways that mediate virtually all developmental signaling, the role of Hedgehog (Hh) proteins is especially prominent in the gut. Two Hh ligands, Sonic (Shh) and Indian (Ihh) are highly expressed early in fetal gut endoderm, from where they signal to the adjacent mesenchyme. Disruption of either gene in mice results in specific but surprisingly limited GI developmental anomalies; because redundancy between the two Hh factors may restrict the scope of defects, we studied mice lacking both Hh factors in gut endoderm. Shh/Ihh-double mutant mice have profoundly abnormal digestive tract development as a result of marked attrition of GI mesenchymal cells. In Specific Aim 1 we propose to define the roles and mechanisms for Hh signaling in early GI development. First, we will dissect the relative importance of Hh signaling in proliferation, survival and differentiation of stomach and intestinal mesenchymal cells, especially with respect to smooth muscle lineages. Second, our observations in mouse embryos that lack all Hh signaling agree with ideas previously proposed in the literature that Hh signals induce expression of the secreted factor Bone morphogenetic protein (BMP)-4 and transcription factors FoxF1 and FoxF2. We propose experiments to determine to what extent these putative downstream targets of Hh signaling are responsible for the global effects on GI organogenesis. The stomach anomalies in Shh/Ihh-double null embryos are identical to those we identified in transgenic mice where the Notch signaling pathway is aberrantly activated in fetal stomach mesenchyme. Other preliminary data also support the novel hypothesis to follow from these findings that Hh signaling in fetal stomach functions in part to restrict mesenchymal Notch activation and hence prevents Notch- induced cell death. Specific Aim 2 outlines a series of in vivo and ex vivo studies to test this hypothesis critically, making particular use of several mouse genetic models to define the nature and extent of interactions between Hh and Notch signaling in GI organogenesis. Although the available experimental models permit incisive experiments in stomach development, similar principles are likely to apply throughout the digestive tract. The studies we propose address important outstanding questions, explore developmental signaling and regulatory mechanisms in molecular detail, and will contribute toward improved understanding and treatment of common GI disorders. PUBLIC HEALTH RELEVANCE. One particular surprise from the last decade of developmental biology research is that complex developmental processes in diverse tissues depend on just a few cell signaling pathways. The hedgehog pathway plays a vital role at many steps in development and homeostasis of the digestive tract and is also implicated in cancers of endodermally derived tissues. The functions and mechanisms of hedgehog signaling in gut development are poorly understood, in spite of their useful implications and applications for human gastrointestinal disorders. This proposal seeks to apply genetic and cell culture tools to elucidate the precise role and mechanisms of hedgehog signaling in genesis of the mouse stomach and intestine
Keywords: Abbreviations; Ablation; Address; Affect; Alimentary Canal; Animal growth regulators, transforming growth factors; Antimorphic mutation; Applied Genetics; Autoregulation; BMP-4; BMP4; Body Tissues; Bone Morphogenetic Proteins; Cancers; Cell Communication and Signaling; Cell Culture Techniques; Cell Death; Cell Signaling; Cell/Tissue, Immunohistochemistry; Cells; Complex; Data; Defect; Development; Developmental Biology; Developmental Process; Digestive Tract; Dominant Negative; Dominant-Negative Mutant; Dominant-Negative Mutation; Embryo; Embryo Development; Embryogenesis; Embryonic; Embryonic Development; Embryonic Tissue; Endoderm; Epithelium; Erinaceidae; Experimental Models; Experimental Models, Other; GI Tract; Gastrointestinal Diseases; Gastrointestinal Diseases and Manifestations; Gastrointestinal Tract; Gastrointestinal tract structure; Gene Expression; Genes; Genetic Models; Genetically Engineered Mouse; Genetics, in situ Hybridization; Hedgehog (Hh) signal transduction pathway; Hedgehogs; Homeostasis; Human; Human, General; IHC; Immunohistochemistry; Immunohistochemistry Staining Method; In Situ Hybridization; Intestinal; Intestines; Intracellular Communication and Signaling; Knock-out; Knockout; Ligands; Literature; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Mesenchymal; Mesenchymas; Mesenchyme; Mesoderm; Mice; Mice, Mutant Strains; Models, Experimental; Molecular; Murine; Mus; Muscle, Involuntary; Muscle, Smooth; Mutant Strains Mice; Nature; Notch Signaling Pathway; Organogenesis; Pathway interactions; Phenocopy; Physiological Homeostasis; Play; Proteins; Proteolysis and Signaling Pathway of Notch; Relative; Relative (related person); Research; Reverse Transcription; Role; Series; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Smooth muscle (tissue); Stomach; Testing; Tissues; Transforming Growth Factors; Transgenic Mice; Tumor Growth Factors; Work; alimentary tract; base; biological signal transduction; bone morphogenetic gene product-4; bone morphogenetic protein 4; bowel; common treatment; digestive canal; early embryonic stage; embryo tissue; experiment; experimental research; experimental study; fetal; fork head protein; forkhead protein; forkhead transcription factors; gastric; gastrointestinal; gastrointestinal disorder; gene product; hedgehog signaling pathway; hh signaling pathway; improved; in situ Hybridization Staining Method; in vivo; malignancy; meetings; mouse mutant; mutant; necrocytosis; neoplasm/cancer; notch; notch protein; notch receptors; novel; pathway; prevent; preventing; public health relevance; research study; smoothened signaling pathway; social role; tissue culture; tool; transcription factor
Relevance: One particular surprise from the last decade of developmental biology research is that complex developmental processes in diverse tissues depend on just a few cell signaling pathways. The hedgehog pathway plays a vital role at many steps in development and homeostasis of the digestive tract and is also implicated in cancers of endodermally derived tissues. The functions and mechanisms of hedgehog signaling in gut development are poorly understood, in spite of their useful implications and applications for human gastrointestinal disorders. This proposal seeks to apply genetic and cell culture tools to elucidate the precise role and mechanisms of hedgehog signaling in genesis of the mouse stomach and intestine
Project start date: 2009-09-01
Project end date: 2013-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
5R01DK081113-02 (2010): $411137
Ramesh A Shivdasani
Dana-farber Cancer Institute
Project start date: 1999-07-01
Project end date: 2013-04-30
Sponsored Links Excellgen http://Excellgen.com
Hedgehog Signaling In Early Development Of The Gastrointestinal Tract
Ramesh A Shivdasani, Associate Professor Of Medicine
Dana-farber Cancer Institute
Grant 1R01DK081113-01A1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: GCMB
Project start date: 2009-09-01
Project end date: 2013-08-31
Gene Expression In The Mammalian Intestine
Ramesh A Shivdasani, Associate Professor Of Medicine
Dana-farber Cancer Institute 44 Binney St Boston, Ma 02115
Grant 3R01DK061139-04S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Project start date: 2002-02-01
Project end date: 2008-01-31
3R01DK061139-04S1 (2007): $102600
NF E2 AND GLOBIN GENE EXPRESSION AND HEMATOPOIESIS
Ramesh A Shivdasani, Associate Professor Of Medicine
Dana-farber Cancer Institute 44 Binney St Boston, Ma 02115
Grant 5K08HL003290-02 from National Heart, Lung, And Blood Institute IRG: ZHL1
Project start date: 1995-05-01
Project end date: 1998-04-30
5K08HL003290-02 (1996): $82836