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Correction of sickle cell disease by homologous recombination in embryonic stem cells
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
that control the globin and other erythroid genes. We have analyzed a 1 Mb region containing the human beta-globin locus and a 600 kb region containing the alpha locus in a variety of erythroid and non-erythroid cell types, including primary tissues. These studies reveal the existence of multiple fetaland adult-erythroid specific DHSs located at considerable distance from known globin control regions. We have also developed a DNA microarray-based approach for mapping tissue-specific DHSs on a genome-wide scale. Combining DNaseI sensitivity studies with a limited number of histone modifications assayed in parallel appears to enable reliable discrimination of promoter-proximal DHSs from distal enhanceror LCR-type sequences. Systematic application of these approaches across a range of erythroid, non-erythroid, and progenitor cell types should enable the production of a comprehensive catalogue of erythroid lineage distal regulatory sequences.
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occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development. References 1) Blazek, E., Mittler, G., Meisterernst, M. The mediator of RNA polymerase II. Chromosoma 113 (2005) 399 –408. 2) Borggrefe, T. The mediator complex in transcriptional regulation. Research Advances in Biological Chemistry 2 (2004)9 –20. 3) Ito, M., Yuan, C. X., Okano, H. J., Darnell, R. B., Roeder, R. G. Involvement of the TRAP220 component of the TRAP/ SMCC coactivator complex in embryonic development and thyroid hormone action. Mol. Cell. 5 (2000) 683– 93. doi:10.1016/j.bcmd.2006.10.133
doi:10.1016/j.bcmd.2006.10.132
122 The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220 Tilman Borggrefe 1, Claudia Waskow 2, Marit Kro¨tschel 1, Dominic van Essen 1, Patrick Rodriguez 1, Boris Guyot 3, Robert G. Roeder 4, Melanie Stumpf 1 1 Department of Cellular and Molecular Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany 2 Department of Immunology, University Clinics Ulm, Germany 3 Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK 4 Laboratory of Biochemistry and Molecular Biology, Rockefeller University, USA The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II [1,2]. Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors [3]. Med1 deficiency causes lethality at embryonic day 11.5 due to defects in heart and placenta development. We show that Med1-deficient 10.5 dpc embryos are anemic, but have normal numbers of hematopoietic progenitor cells. Med1deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-Es) and colony-forming units (CFU-Es) but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. Expression of endogenous GATA-1 target genes is severely affected in Med1-deficient progenitor cells. Moreover, in transcription assays Med1 deficiency leads to a defect in GATA-1mediated transactivation. In chromatin immunoprecipitation experiments we find Mediator components at GATA-1
123 Correction of sickle cell disease by homologous recombination in embryonic stem cells Chiao-Wang Sun, Li-Chen Wu, Thomas M. Ryan, Kevin M. Pawlik, Jinxiang Ren, Tim M. Townes Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry, Birmingham, AL 35294, USA Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing anti-sickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (hS) with a normal copy of the gene (hA). We recently derived embryonic stem (ES) cells from a novel knockin mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human hS-globin gene with a human hA-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematological values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis. doi:10.1016/j.bcmd.2006.10.134
that control the globin and other erythroid genes. We have analyzed a 1 Mb region containing the human beta-globin locus and a 600 kb region containing the alpha locus in a variety of erythroid and non-erythroid cell types, including primary tissues. These studies reveal the existence of multiple fetaland adult-erythroid specific DHSs located at considerable distance from known globin control regions. We have also developed a DNA microarray-based approach for mapping tissue-specific DHSs on a genome-wide scale. Combining DNaseI sensitivity studies with a limited number of histone modifications assayed in parallel appears to enable reliable discrimination of promoter-proximal DHSs from distal enhanceror LCR-type sequences. Systematic application of these approaches across a range of erythroid, non-erythroid, and progenitor cell types should enable the production of a comprehensive catalogue of erythroid lineage distal regulatory sequences.
179
occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development. References 1) Blazek, E., Mittler, G., Meisterernst, M. The mediator of RNA polymerase II. Chromosoma 113 (2005) 399 –408. 2) Borggrefe, T. The mediator complex in transcriptional regulation. Research Advances in Biological Chemistry 2 (2004)9 –20. 3) Ito, M., Yuan, C. X., Okano, H. J., Darnell, R. B., Roeder, R. G. Involvement of the TRAP220 component of the TRAP/ SMCC coactivator complex in embryonic development and thyroid hormone action. Mol. Cell. 5 (2000) 683– 93. doi:10.1016/j.bcmd.2006.10.133
doi:10.1016/j.bcmd.2006.10.132
122 The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220 Tilman Borggrefe 1, Claudia Waskow 2, Marit Kro¨tschel 1, Dominic van Essen 1, Patrick Rodriguez 1, Boris Guyot 3, Robert G. Roeder 4, Melanie Stumpf 1 1 Department of Cellular and Molecular Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany 2 Department of Immunology, University Clinics Ulm, Germany 3 Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK 4 Laboratory of Biochemistry and Molecular Biology, Rockefeller University, USA The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II [1,2]. Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors [3]. Med1 deficiency causes lethality at embryonic day 11.5 due to defects in heart and placenta development. We show that Med1-deficient 10.5 dpc embryos are anemic, but have normal numbers of hematopoietic progenitor cells. Med1deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-Es) and colony-forming units (CFU-Es) but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. Expression of endogenous GATA-1 target genes is severely affected in Med1-deficient progenitor cells. Moreover, in transcription assays Med1 deficiency leads to a defect in GATA-1mediated transactivation. In chromatin immunoprecipitation experiments we find Mediator components at GATA-1
123 Correction of sickle cell disease by homologous recombination in embryonic stem cells Chiao-Wang Sun, Li-Chen Wu, Thomas M. Ryan, Kevin M. Pawlik, Jinxiang Ren, Tim M. Townes Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry, Birmingham, AL 35294, USA Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing anti-sickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (hS) with a normal copy of the gene (hA). We recently derived embryonic stem (ES) cells from a novel knockin mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human hS-globin gene with a human hA-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematological values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis. doi:10.1016/j.bcmd.2006.10.134