- Email: [email protected]
HbVar database for human hemoglobin variants and thalassemia mutations
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
indicating that both the primitive erythroid and megakaryocyte lineages emerge from hemangioblast precursors during gastrulation. A transient wave of primitive-MEP was found exclusively in the yolk sac between E7.25 and E9.0, indicating that primitive hematopoiesis is bilineage in nature. The association of MEP with primitive erythroid progenitors prompted us to explore the relationship of the erythroid and megakaryocyte lineages as BFU-E subsequently emerge in the yolk sac, using the pan-erythroid Ter119 antibody to track erythroid progenitors between E9.0 and E11.5. Initially, both Meg-CFC and definitive-MEP were localized exclusively in the yolk sac. As development proceeded, these progenitors expanded in the yolk sac and were found in increasing numbers in the circulation. At 10.5, definitive-MEP peaked in the yolk sac and small numbers of Meg-CFC were detected within the liver. By E11.5, the liver became the predominant site of definitive erythroid and megakaryocyte progenitor expansion while the number of Meg-CFC and definitive-MEP decreased in the yolk sac, suggesting that yolk sac-derived erythroid and megakaryocyte progenitors are among the first hematopoietic elements to engraft the newly forming liver rudiment. The findings of Li et al. (Nature Genetics 63:613, 2005), that forced expression of truncated GATA-1 induces the proliferation of megakaryocyte cells from the E9.5 yolk sac and E12.5 fetal liver, suggest that the Meg-CFC and definitive-MEP described here may be the murine equivalent of the target cell responsible for TMD and acute megakaryocytic leukemia in children with Down syndrome. We conclude that the erythroid and megakaryocyte lineages are initially derived from hemangioblast precursors and that the primitive and definitive erythroid lineages are each associated with the megakaryocyte lineage through a common bipotential megakaryocyte/erythroid progenitor.
doi:10.1016/j.bcmd.2006.10.107
97 Programming of HSCs during development Aldo Ciau-Uitz, Phil Pinheiro, Claire Fernandez, Tariq Enver, Roger Patient MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Headington, Oxford, UK It is becoming increasingly clear that the switch from embryonic globin gene expression in the developing embryo is a consequence of red cell production from a developmentally distinct lineage of cells. The adult globin expressing lineage derives from haematopoietic stem cells (HSCs), which arise in close association with the dorsal aorta. We have defined roles for several signalling pathways in the specification of HSCs and are detailing the unfolding of the nuclear programme by determining the functional hierarchies amongst the haematopoietic transcription factors.
167
We will present novel observations on the roles of Runx1 and Tel1. doi:10.1016/j.bcmd.2006.10.108
98 HbVar database for human hemoglobin variants and thalassemia mutations R.C. Hardison 1, B. Giardine 2, C. Riemer 2, W. Miller 2, D.H.K. Chui 3, H. Wajcman 4, G.P. Patrinos 5 1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA 2 Department of Computer Science and Engineering, The Pennsylvania State University, University Park, PA, USA 3 Departments of Medicine and Pathology, Boston University School of Medicine, Boston, MA, USA 4 INSERM-U654 Bases Mole´culaires et Cellulaires des Maladies Genetiques, Hoˆpital Henri Mondor, Cre´teil, France 5 Erasmus MC, Faculty of Medicine and Health Sciences, MGC-Department of Cell Biology and Genetics, Rotterdam, The Netherlands Mutation databases are continuously updated and quality controlled repositories for collection, documentation, storage and organization of the enormous amount of genomic data, related to inherited disorders. HbVar (http://globin.cse.psu.edu/ hbvar) is a relational locus-specific database (LSDB) developed in 2001 by a multi-center academic effort to provide timely information on the genomic sequence changes leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Database records include extensive phenotypic descriptions, biochemical and hematological effects, associated pathology and ethnic occurrence, accompanied by mutation frequencies and references. In addition to the regular updates to entries, we report significant advances and updates, which can be also useful not only for HbVar users but also for other LSDB development and curation in general. The query page has been reconstructed to become user-friendlier and at the same time to include more functionalities, automatic routines to identify known single nucleotide polymorphisms in the human a- and h-globin loci have been implemented and more population specific data have been added and updated for a-, h- and y-thalassemias for several population groups. In addition, XPRbase (http://www.goldenhelix.org/xprbase), an independent flat-file database, has been developed and linked to the main HbVar web page to provide a succinct listing of several experimental protocols available for globin gene mutation screening. Finally, a step towards integrating the available resources in the Globin Gene Server is PHENCODE. As LSDBs are a key connection between the abundance of genomic information and clinically important issues, HbVar and GenPhen databases have been adapted to complete a path from genome sequence to functional analysis, via ENCODE (http://www.genome.gov/10005107) to human mutations (HbVar) and on to clinical phenotypes of groups of patients (GenPhen) and back. We anticipate that those updates will
168
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
significantly ameliorate the database profile and quality of information provided and is anticipated to increase the already high impact of HbVar database. doi:10.1016/j.bcmd.2006.10.109
99 BMP4-dependent signals regulate the expansion of a specialized population of erythroid progenitors during times of erythropoietic stress Robert F. Paulson, John Perry, Omid Harandi, Prashanth Porayette, Shailaja Hegde, Dai Chen Wu, Lei Shi Department of Veterinary and Biomedical Sciences, The Center for Molecular Immunology and Infectious Disease, The Pennsylvania State University, University Park, PA 16802, USA Bone marrow erythropoiesis is primarily homeostatic, constantly producing new erythrocytes throughout adult life. The situation is dramatically different during embryogenesis and in response to acute anemia in adults. At these times expansive or stress erythropoiesis occurs. Our initial work showed that BMP4 regulates the erythroid response to acute anemia by inducing the rapid expansion in the spleen of a specialized population of erythroid progenitors termed stress-BFU-E. These progenitors are distinct from bone marrow steady state BFU-E in that they are able to rapidly produce large numbers erythrocytes. BMP4 signaling is required in vivo for the expansion of stress BFU-E, however in vitro, BMP4 alone cannot recapitulate the expansion of stress BFU-E observed in vivo suggesting that other signals are required. Here we report that mutation of the Kit receptor, results in a severe defect in the expansion of stress BFU-E indicating a role for the Kit/SCF signaling pathway in splenic stress erythropoiesis. In vitro analysis showed that BMP4 and SCF are necessary for the expansion of stress BFU-E, but only when spleen cells were cultured in BMP4 + SCF at low oxygen concentrations did we recapitulate the expansion of stress BFUE observed in vivo. Taken together these data demonstrate that SCF and hypoxia synergize with BMP4 to promote the expansion and differentiation of stress BFU-E during the recovery from acute anemia. In addition, we have expanded our analysis of stress erythropoiesis to include two other instances of expansive erythropoiesis, fetal liver erythropoiesis and erythropoiesis induced by Friend erythroleukemia virus. In both of these cases, BMP4-dependent signaling is required. Analysis of fetal liver erythropoiesis showed that BMP4 drives the initial expansion of a population of BFU-E that exhibits properties similar to adult stress BFU-E. Mutations that affect BMP4 signaling specifically affect the early expansion of stress-like BFU-E in the fetal liver and lead to severe anemia during early fetal development. We have also observed that the rapid erythropoiesis induced by Friend virus infection requires the BMP4-dependent stress erythropoiesis pathway. Mutations that affect BMP4 signaling render mice resistant to Friend virus. Friend virus infection induces the expression of BMP4 in the spleen, which leads to the expansion of stress BFU-E, which
are the target cells for Friend virus. Our data indicate that Friend virus exploits the BMP4-dependent expansion of stress BFU-E to amplify target cells for the virus, which leads to rapid splenomegaly and polycythemia. The analysis of stress erythropoiesis in these three systems clearly demonstrates that BMP4 is the key signal that regulates stress erythropoiesis. doi:10.1016/j.bcmd.2006.10.110
100 Erythroid Kruppel-like factor regulates the G1 Cdk inhibitor p18 Michael R. Tallack, Janelle R. Keys, Andrew C. Perkins Institute for Molecular Bioscience, Brisbane, QLD, Australia Erythroid Kruppel-like Factor (EKLF) is a zinc finger transcription factor that is expressed specifically in erythrocytes throughout development. Its major function is the activation of h-globin gene expression, by binding to CACCC box motifs, however further roles in erythrocyte development appear likely. A transcriptional profiling experiment comparing the global gene expression in EKLF-null and wild-type erythrocytes has identified many differentially expressed genes. The Cyclindependent kinase (Cdk) inhibitor p18 was identified as a potential EKLF target gene, and found to be down-regulated in EKLF-null mice as confirmed by real-time PCR. The p18 protein functions as an inhibitor of Cdk4 and Cdk6 activity during early G1 phase of the cell cycle to control its progression. The search throughout the p18 gene locus for phylogenetically conserved extended CACC box elements (CCNCNCCC) found two closely associated CACC sites ¨ 1 kb upstream of the transcriptional start site. We show EKLF binding to these sites by gel shift assay and have demonstrated that these sites are capable of driving EKLF-dependent transcription in luciferase reporter assays. We also show by chromatin immunoprecipitation (ChIP) assay that this region of the p18 promoter is specifically occupied by EKLF in vivo. These results suggest that EKLF acts to control the switch from proliferation to differentiation in erythrocytes by regulating p18 gene expression and thereby controlling passage through the G1 – S transition. doi:10.1016/j.bcmd.2006.10.111
101 The transcriptional factor, IKAROS, plays a key role in ; –B globin gene switching J.R. Keys 1, P. Papathanasiou 2, C.C. Goodnow 2, A.C. Perkins 1 1 Institute for Molecular Bioscience, University of Queensland, QLD, Australia 2 John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia The zinc finger transcription factor Ikaros plays a number of roles in haematopoiesis. A mouse strain, IkarosPlastic, with a
indicating that both the primitive erythroid and megakaryocyte lineages emerge from hemangioblast precursors during gastrulation. A transient wave of primitive-MEP was found exclusively in the yolk sac between E7.25 and E9.0, indicating that primitive hematopoiesis is bilineage in nature. The association of MEP with primitive erythroid progenitors prompted us to explore the relationship of the erythroid and megakaryocyte lineages as BFU-E subsequently emerge in the yolk sac, using the pan-erythroid Ter119 antibody to track erythroid progenitors between E9.0 and E11.5. Initially, both Meg-CFC and definitive-MEP were localized exclusively in the yolk sac. As development proceeded, these progenitors expanded in the yolk sac and were found in increasing numbers in the circulation. At 10.5, definitive-MEP peaked in the yolk sac and small numbers of Meg-CFC were detected within the liver. By E11.5, the liver became the predominant site of definitive erythroid and megakaryocyte progenitor expansion while the number of Meg-CFC and definitive-MEP decreased in the yolk sac, suggesting that yolk sac-derived erythroid and megakaryocyte progenitors are among the first hematopoietic elements to engraft the newly forming liver rudiment. The findings of Li et al. (Nature Genetics 63:613, 2005), that forced expression of truncated GATA-1 induces the proliferation of megakaryocyte cells from the E9.5 yolk sac and E12.5 fetal liver, suggest that the Meg-CFC and definitive-MEP described here may be the murine equivalent of the target cell responsible for TMD and acute megakaryocytic leukemia in children with Down syndrome. We conclude that the erythroid and megakaryocyte lineages are initially derived from hemangioblast precursors and that the primitive and definitive erythroid lineages are each associated with the megakaryocyte lineage through a common bipotential megakaryocyte/erythroid progenitor.
doi:10.1016/j.bcmd.2006.10.107
97 Programming of HSCs during development Aldo Ciau-Uitz, Phil Pinheiro, Claire Fernandez, Tariq Enver, Roger Patient MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Headington, Oxford, UK It is becoming increasingly clear that the switch from embryonic globin gene expression in the developing embryo is a consequence of red cell production from a developmentally distinct lineage of cells. The adult globin expressing lineage derives from haematopoietic stem cells (HSCs), which arise in close association with the dorsal aorta. We have defined roles for several signalling pathways in the specification of HSCs and are detailing the unfolding of the nuclear programme by determining the functional hierarchies amongst the haematopoietic transcription factors.
167
We will present novel observations on the roles of Runx1 and Tel1. doi:10.1016/j.bcmd.2006.10.108
98 HbVar database for human hemoglobin variants and thalassemia mutations R.C. Hardison 1, B. Giardine 2, C. Riemer 2, W. Miller 2, D.H.K. Chui 3, H. Wajcman 4, G.P. Patrinos 5 1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA 2 Department of Computer Science and Engineering, The Pennsylvania State University, University Park, PA, USA 3 Departments of Medicine and Pathology, Boston University School of Medicine, Boston, MA, USA 4 INSERM-U654 Bases Mole´culaires et Cellulaires des Maladies Genetiques, Hoˆpital Henri Mondor, Cre´teil, France 5 Erasmus MC, Faculty of Medicine and Health Sciences, MGC-Department of Cell Biology and Genetics, Rotterdam, The Netherlands Mutation databases are continuously updated and quality controlled repositories for collection, documentation, storage and organization of the enormous amount of genomic data, related to inherited disorders. HbVar (http://globin.cse.psu.edu/ hbvar) is a relational locus-specific database (LSDB) developed in 2001 by a multi-center academic effort to provide timely information on the genomic sequence changes leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Database records include extensive phenotypic descriptions, biochemical and hematological effects, associated pathology and ethnic occurrence, accompanied by mutation frequencies and references. In addition to the regular updates to entries, we report significant advances and updates, which can be also useful not only for HbVar users but also for other LSDB development and curation in general. The query page has been reconstructed to become user-friendlier and at the same time to include more functionalities, automatic routines to identify known single nucleotide polymorphisms in the human a- and h-globin loci have been implemented and more population specific data have been added and updated for a-, h- and y-thalassemias for several population groups. In addition, XPRbase (http://www.goldenhelix.org/xprbase), an independent flat-file database, has been developed and linked to the main HbVar web page to provide a succinct listing of several experimental protocols available for globin gene mutation screening. Finally, a step towards integrating the available resources in the Globin Gene Server is PHENCODE. As LSDBs are a key connection between the abundance of genomic information and clinically important issues, HbVar and GenPhen databases have been adapted to complete a path from genome sequence to functional analysis, via ENCODE (http://www.genome.gov/10005107) to human mutations (HbVar) and on to clinical phenotypes of groups of patients (GenPhen) and back. We anticipate that those updates will
168
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
significantly ameliorate the database profile and quality of information provided and is anticipated to increase the already high impact of HbVar database. doi:10.1016/j.bcmd.2006.10.109
99 BMP4-dependent signals regulate the expansion of a specialized population of erythroid progenitors during times of erythropoietic stress Robert F. Paulson, John Perry, Omid Harandi, Prashanth Porayette, Shailaja Hegde, Dai Chen Wu, Lei Shi Department of Veterinary and Biomedical Sciences, The Center for Molecular Immunology and Infectious Disease, The Pennsylvania State University, University Park, PA 16802, USA Bone marrow erythropoiesis is primarily homeostatic, constantly producing new erythrocytes throughout adult life. The situation is dramatically different during embryogenesis and in response to acute anemia in adults. At these times expansive or stress erythropoiesis occurs. Our initial work showed that BMP4 regulates the erythroid response to acute anemia by inducing the rapid expansion in the spleen of a specialized population of erythroid progenitors termed stress-BFU-E. These progenitors are distinct from bone marrow steady state BFU-E in that they are able to rapidly produce large numbers erythrocytes. BMP4 signaling is required in vivo for the expansion of stress BFU-E, however in vitro, BMP4 alone cannot recapitulate the expansion of stress BFU-E observed in vivo suggesting that other signals are required. Here we report that mutation of the Kit receptor, results in a severe defect in the expansion of stress BFU-E indicating a role for the Kit/SCF signaling pathway in splenic stress erythropoiesis. In vitro analysis showed that BMP4 and SCF are necessary for the expansion of stress BFU-E, but only when spleen cells were cultured in BMP4 + SCF at low oxygen concentrations did we recapitulate the expansion of stress BFUE observed in vivo. Taken together these data demonstrate that SCF and hypoxia synergize with BMP4 to promote the expansion and differentiation of stress BFU-E during the recovery from acute anemia. In addition, we have expanded our analysis of stress erythropoiesis to include two other instances of expansive erythropoiesis, fetal liver erythropoiesis and erythropoiesis induced by Friend erythroleukemia virus. In both of these cases, BMP4-dependent signaling is required. Analysis of fetal liver erythropoiesis showed that BMP4 drives the initial expansion of a population of BFU-E that exhibits properties similar to adult stress BFU-E. Mutations that affect BMP4 signaling specifically affect the early expansion of stress-like BFU-E in the fetal liver and lead to severe anemia during early fetal development. We have also observed that the rapid erythropoiesis induced by Friend virus infection requires the BMP4-dependent stress erythropoiesis pathway. Mutations that affect BMP4 signaling render mice resistant to Friend virus. Friend virus infection induces the expression of BMP4 in the spleen, which leads to the expansion of stress BFU-E, which
are the target cells for Friend virus. Our data indicate that Friend virus exploits the BMP4-dependent expansion of stress BFU-E to amplify target cells for the virus, which leads to rapid splenomegaly and polycythemia. The analysis of stress erythropoiesis in these three systems clearly demonstrates that BMP4 is the key signal that regulates stress erythropoiesis. doi:10.1016/j.bcmd.2006.10.110
100 Erythroid Kruppel-like factor regulates the G1 Cdk inhibitor p18 Michael R. Tallack, Janelle R. Keys, Andrew C. Perkins Institute for Molecular Bioscience, Brisbane, QLD, Australia Erythroid Kruppel-like Factor (EKLF) is a zinc finger transcription factor that is expressed specifically in erythrocytes throughout development. Its major function is the activation of h-globin gene expression, by binding to CACCC box motifs, however further roles in erythrocyte development appear likely. A transcriptional profiling experiment comparing the global gene expression in EKLF-null and wild-type erythrocytes has identified many differentially expressed genes. The Cyclindependent kinase (Cdk) inhibitor p18 was identified as a potential EKLF target gene, and found to be down-regulated in EKLF-null mice as confirmed by real-time PCR. The p18 protein functions as an inhibitor of Cdk4 and Cdk6 activity during early G1 phase of the cell cycle to control its progression. The search throughout the p18 gene locus for phylogenetically conserved extended CACC box elements (CCNCNCCC) found two closely associated CACC sites ¨ 1 kb upstream of the transcriptional start site. We show EKLF binding to these sites by gel shift assay and have demonstrated that these sites are capable of driving EKLF-dependent transcription in luciferase reporter assays. We also show by chromatin immunoprecipitation (ChIP) assay that this region of the p18 promoter is specifically occupied by EKLF in vivo. These results suggest that EKLF acts to control the switch from proliferation to differentiation in erythrocytes by regulating p18 gene expression and thereby controlling passage through the G1 – S transition. doi:10.1016/j.bcmd.2006.10.111
101 The transcriptional factor, IKAROS, plays a key role in ; –B globin gene switching J.R. Keys 1, P. Papathanasiou 2, C.C. Goodnow 2, A.C. Perkins 1 1 Institute for Molecular Bioscience, University of Queensland, QLD, Australia 2 John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia The zinc finger transcription factor Ikaros plays a number of roles in haematopoiesis. A mouse strain, IkarosPlastic, with a