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TAL-1 and GATA-1 coupled cell proliferation and differentiation during terminal erythroid differentiation
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
focus on different aspects of red cell biology. The first screen aims to identify ENU induced mutations that result in enhanced expression of g-globin genes. For this screen we have employed transgenic mice carrying the human h-globin locus on a yeast artificial chromosome. To this date we have identified five lines with elevated HbF levels. Outbreeding of these animals found the phenotype to be heritable according to Mendelian ratios. We are now in the process of mapping the mutated chromosomal loci and identifying potential candidate genes. In our second ENU screen (RBC screen) we focus on identifying dominant and recessive mutations that effect red blood cell production and maturation. G1 mice are screened by analysis of their full blood count at weaning, and animals with an abnormal mean cell volume (MCV) and normal white cell and platelet counts prioritised. We have recovered eight independent lines with markedly abnormal red cell indices, in which the phenotype was heritable. Five of these lines displayed a reduced MCV that lay more than three standard deviations outside the population mean. This presentation focuses on one line (RBC2) with a defect in the membrane skeleton resulting in severe haemolytic anaemia. Mice homozygous for the mutation are profoundly anaemic and jaundiced at birth with massive splenomegaly, and the majority die before weaning. The remaining animals rapidly developed iron overload and had a life span of only 5 –8 weeks. Sequencing of their erythroid ankyrin-1 (Ank1) gene identified a novel splice mutation, which results in a frame shift that introduces a stop 96 codons downstream, and predicted a 194 kDa ankyrin protein. Analysis of RBC ghosts from mutant mice revealed almost a complete absence of Ank1, co-incident with almost total loss of a- and h-spectrin, and band 4.2. The mutant animals displayed increased resistance to malaria parasites. These features make this Ank1-deficient mouse line an ideal model to study ankyrin-dependent blood disorders. doi:10.1016/j.bcmd.2006.10.123
113 Comparing genetic profiles of embryonic day 9 (E9) mouse yolk sac erythroid and erythroid and epithelial cells isolated by microdissection Latasha C. Redmond 1, Jack L. Haar 2, Catherine I. Dumur 3, Kellie J. Archer 4, Priyadarshi Basu 1, Joyce A. Lloyd 1,5 1 Department of Human Genetics, Virginia Commonwealth University, Richmond, VA, USA 2 Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA, USA 3 Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA 4 Department of Biostatistics, Virginia Commonwealth University, Richmond, VA, USA 5 Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA Erythropoiesis in the embryonic day 9 (E9) mouse yolk sac produces cells having embryonic globin, followed by hepatic and
175
myeloid erythropoiesis resulting in erythrocytes with fetal and adult globins. The purpose of this study was to generate a genetic profile of mouse erythroid development in order to identify genes that are essential for embryonic erythropoiesis. Yolk sacs from E9 mice were cryoprotected with 20% sucrose in PBS. Laser capture microdissection (LCM) was used to isolate primitive erythroid and epithelial cells from frozen sections of the yolk sac and highquality RNA was obtained. The RNA samples were amplified using a two-round linear amplification protocol, labeled for hybridization to the microarrays, and differentially expressed genes were determined using the Significance S-score method. Seventy-five genes are expressed higher in erythroid than in epithelial cells, and five of these genes were of unknown function. Many known erythroid-specific genes including globin, red cell skeletal proteins including glycophorin A, enzymes involved in heme synthesis, and transcription factors including EKLF/KLF1 are expressed significantly higher in erythroid than epithelial cells. Ingenuity Pathway Analysis indicates that several of the upregulated genes cluster in a highly significant gene network that contains the embryonic globins, EKLF/KLF1 and LMO2, one of the few proteins already known to be required for embryonic erythropoiesis. Quantitative RT–PCR verified that LMO2, and glycophorin A mRNAs are expressed higher in erythroid than in epithelial cells. The results indicate that genes important for primitive erythropoiesis can be identified with this method. This will have clinical implications for treating h-hemoglobinopathies. doi:10.1016/j.bcmd.2006.10.124
114 TAL-1 and GATA-1 coupled cell proliferation and differentiation during terminal erythroid differentiation Zahra Kadri 1, Nicolas Goardon 1, Ritsuko Shimizu 2, Osamu Ohneda 2, Tran Hoang 4, Philippe Leboulch 3, Sylvie Gisselbrecht 1, Masayuki Yamamoto 2, Stany Chretien 1,3, Paul Henri Romeo 1 1 Institut Cochin, De´partement d’He´matologie, INSERM U567, CNRS UMR 8104, Universite´ Paris V, Paris, France 2 Department of Molecular and Developmental Biology, Center for TARA, ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan 3 Institute of Research in Immunology and Cancer, University of Montreal, Montreal, Quebec, Canada 4 Genetics Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 5 INSERM U733, Hoˆpital Saint Louis, Paris, France With more than a hundred billion red blood cells generated every day, the erythroid lineage has the largest quantitative output of cell production in adult mammals. This impressive capability requires a pattern of cell proliferation closely related to that of embryonic cells followed by ultimate inhibition of cell division, as terminal erythroid differentiation is completed. Yet, the putative molecular pathways that coordinate this cell proliferation arrest and differentiation remain obscure. We will show data that indicate that TAL-1 and GATA-1, two transcrip-
176
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
tion factors essential for erythroid differentiation regulate the cell cycle during terminal erythroid differentiation. During the late phases of erythropoiesis, TAL-1 directly regulated the gene expression of two essential regulators of erythroid cell proliferation, Gfi-1b and the cyclin-dependent kinase inhibitor and G1 checkpoint regulator p21Cip. This transcriptional regulation is mediated by the association/dissociation of TAL-1 bound to the promoters of these two genes and ETO2, a known transcriptional repressor. We will also show that the differentiation factor GATA-1 directly interacted with the retinoblastoma (pRb) protein during the late stages of erythropoiesis. This association required the canonical LXCXE motif localized between amino acids 81 and 85 of GATA-1. This sequence is conserved during evolution, is not found in the other members of the GATA family and is absent in the short form of GATA-1 expressed in the acute megakaryoblastic leukemia of Down syndrome. The inhibition of cellular proliferation induced by GATA-1 over-expression was inefficient in pRb negative cell lines or when pRb was knocked down by specific shRNA. Using a GATA-1 protein mutated on the LXCXE motif (GATA-1Rb ), we showed that (i) the LXCXE motif was necessary for GATA-1/pRb association, (ii) the over-expression of GATA-1Rb did not inhibit cellular proliferation whereas GATA-1 over-expression did, and (iii) the expression of GATA-1Rb in a GATA-1-deficient erythroid cell line decreased the number of benzidine positive cells and increased the immature erythroid fraction. Finally, endogenous level expression of GATA-1Rb with GATA-1 HRD vector in the GATA-1.05 genotype mice resulted in a partial rescue of GATA-1.05 embryos from the early lethality at E11.5, but the embryos then exhibited severe anemia and died by E15.5. Analysis of the E12.5 fetal liver of these GATA-1Rb rescued mice showed an increased number of immature erythroid cells and a decreased number of mature erythroid cells. These results indicate that the GATA-1 and TAL-1 coordinate the expression of erythroid specific markers and the inhibition of cell division that occurs during the late stages of erythropoiesis. doi:10.1016/j.bcmd.2006.10.125
115 Activation of the LMO2 gene in human lymphoid cells by targeted insertion of a single retroviral LTR expression cassette into the first intron Byoung Y. Ryu 1, John T. Gray 1, David M. Bodine 2, Arthur W. Nienhuis 1 1 Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 2 Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD, USA The pathogenic activation of the LMO2 proto-oncogene contributed to leukemia in two children otherwise successfully treated for Severe Combined Immunodeficiency (SCID) in a gene therapy trial (Science 302:415–419, 2003). We have reproduced the retroviral vector insertion into the first intron of the LMO2
gene that occurred in one of the SCID patients in a human lympholeukemia cell line (Jurkat) using recombinant adenoassociated viral (rAAV) vector mediated gene targeting. The rAAV-LMO2 vector was constructed as follows. First, 1.3 kb fragments containing LMO2 intron sequences from upstream and downstream of the retroviral insertion site in the SCID patient were amplified by PCR from human genomic DNA and sequenced. Next, a gene cassette was designed to have the GFP coding sequences under the control of a single retroviral LTR that was used in the gene therapy trial followed by a polyadenylation signal, which in turn flanked by loxP sequences. This floxed LTR–GFP cassette was then placed in a reverse orientation between the 5V and 3V segments from LMO2 intron 1 and the fragments were cloned between AAV ITRs to derive the rAAV targeting vector. The Jurkat T cell line was transduced with rAAV–LMO2 particles and GFP expressing cells were selected by FACS at 9 days post-transduction and clones derived from single cells were recovered. The GFP-positive clones were screened for targeting into the LMO2 locus by PCR and Southern hybridization. Four clones showed the predicted bands in addition to wild-type bands indicating successful gene targeting into one of the LMO2 alleles. Next, LMO2 expression was compared to that in control Jurkat cells by real-time quantitative RT–PCR using the comparative CT method. Expression of LMO2 mRNA was at a very low level in untransduced Jurkat cells but was highly upregulated in all 4 clones ranging from an 1100- to 3500-fold increase. LMO2 protein was expressed in 4 clones but not in untransduced Jurkat consistent with real-time PCR results. Then, floxed DsRed expression cassette was constructed and used for Cre/loxP exchange in LMO2-targeted Jurkat clone. GFP expressing Jurkat cells were transduced with rAAV-Cre and transfected with DsRed cassette on following day. Single cell clones were isolated, expanded, and screened for DsRed expression without GFP expression. Southern hybridization was performed to confirm cassette exchange and four clones appeared to have cassette exchanged. Thus this system should prove useful for evaluating the relative safety of specific retroviral vector designs with respect to the potential for proto-oncogene activation. Currently, an exchange cassette containing globin LCR has been constructed and undergone Cre/loxP exchange. doi:10.1016/j.bcmd.2006.10.126
116 The expanding application of lentivirus-mediated erythroid-specific gene therapy Michel Sadelain, Selda Samakoglu, Alex Chang, Leszek Lisowski Memorial Sloan-Kettering Cancer Center, New York, NY, USA Recombinant lentiviruses are stable vectors that mediate efficient transfer of complex transcription units including large genomic fragments. We previously characterized in detail the vector termed TNS9, which encodes the human beta-globin gene, including an extended promoter both introns and both proximal enhancers, as well as large HS2, HS3 and HS4
focus on different aspects of red cell biology. The first screen aims to identify ENU induced mutations that result in enhanced expression of g-globin genes. For this screen we have employed transgenic mice carrying the human h-globin locus on a yeast artificial chromosome. To this date we have identified five lines with elevated HbF levels. Outbreeding of these animals found the phenotype to be heritable according to Mendelian ratios. We are now in the process of mapping the mutated chromosomal loci and identifying potential candidate genes. In our second ENU screen (RBC screen) we focus on identifying dominant and recessive mutations that effect red blood cell production and maturation. G1 mice are screened by analysis of their full blood count at weaning, and animals with an abnormal mean cell volume (MCV) and normal white cell and platelet counts prioritised. We have recovered eight independent lines with markedly abnormal red cell indices, in which the phenotype was heritable. Five of these lines displayed a reduced MCV that lay more than three standard deviations outside the population mean. This presentation focuses on one line (RBC2) with a defect in the membrane skeleton resulting in severe haemolytic anaemia. Mice homozygous for the mutation are profoundly anaemic and jaundiced at birth with massive splenomegaly, and the majority die before weaning. The remaining animals rapidly developed iron overload and had a life span of only 5 –8 weeks. Sequencing of their erythroid ankyrin-1 (Ank1) gene identified a novel splice mutation, which results in a frame shift that introduces a stop 96 codons downstream, and predicted a 194 kDa ankyrin protein. Analysis of RBC ghosts from mutant mice revealed almost a complete absence of Ank1, co-incident with almost total loss of a- and h-spectrin, and band 4.2. The mutant animals displayed increased resistance to malaria parasites. These features make this Ank1-deficient mouse line an ideal model to study ankyrin-dependent blood disorders. doi:10.1016/j.bcmd.2006.10.123
113 Comparing genetic profiles of embryonic day 9 (E9) mouse yolk sac erythroid and erythroid and epithelial cells isolated by microdissection Latasha C. Redmond 1, Jack L. Haar 2, Catherine I. Dumur 3, Kellie J. Archer 4, Priyadarshi Basu 1, Joyce A. Lloyd 1,5 1 Department of Human Genetics, Virginia Commonwealth University, Richmond, VA, USA 2 Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA, USA 3 Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA 4 Department of Biostatistics, Virginia Commonwealth University, Richmond, VA, USA 5 Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA Erythropoiesis in the embryonic day 9 (E9) mouse yolk sac produces cells having embryonic globin, followed by hepatic and
175
myeloid erythropoiesis resulting in erythrocytes with fetal and adult globins. The purpose of this study was to generate a genetic profile of mouse erythroid development in order to identify genes that are essential for embryonic erythropoiesis. Yolk sacs from E9 mice were cryoprotected with 20% sucrose in PBS. Laser capture microdissection (LCM) was used to isolate primitive erythroid and epithelial cells from frozen sections of the yolk sac and highquality RNA was obtained. The RNA samples were amplified using a two-round linear amplification protocol, labeled for hybridization to the microarrays, and differentially expressed genes were determined using the Significance S-score method. Seventy-five genes are expressed higher in erythroid than in epithelial cells, and five of these genes were of unknown function. Many known erythroid-specific genes including globin, red cell skeletal proteins including glycophorin A, enzymes involved in heme synthesis, and transcription factors including EKLF/KLF1 are expressed significantly higher in erythroid than epithelial cells. Ingenuity Pathway Analysis indicates that several of the upregulated genes cluster in a highly significant gene network that contains the embryonic globins, EKLF/KLF1 and LMO2, one of the few proteins already known to be required for embryonic erythropoiesis. Quantitative RT–PCR verified that LMO2, and glycophorin A mRNAs are expressed higher in erythroid than in epithelial cells. The results indicate that genes important for primitive erythropoiesis can be identified with this method. This will have clinical implications for treating h-hemoglobinopathies. doi:10.1016/j.bcmd.2006.10.124
114 TAL-1 and GATA-1 coupled cell proliferation and differentiation during terminal erythroid differentiation Zahra Kadri 1, Nicolas Goardon 1, Ritsuko Shimizu 2, Osamu Ohneda 2, Tran Hoang 4, Philippe Leboulch 3, Sylvie Gisselbrecht 1, Masayuki Yamamoto 2, Stany Chretien 1,3, Paul Henri Romeo 1 1 Institut Cochin, De´partement d’He´matologie, INSERM U567, CNRS UMR 8104, Universite´ Paris V, Paris, France 2 Department of Molecular and Developmental Biology, Center for TARA, ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan 3 Institute of Research in Immunology and Cancer, University of Montreal, Montreal, Quebec, Canada 4 Genetics Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 5 INSERM U733, Hoˆpital Saint Louis, Paris, France With more than a hundred billion red blood cells generated every day, the erythroid lineage has the largest quantitative output of cell production in adult mammals. This impressive capability requires a pattern of cell proliferation closely related to that of embryonic cells followed by ultimate inhibition of cell division, as terminal erythroid differentiation is completed. Yet, the putative molecular pathways that coordinate this cell proliferation arrest and differentiation remain obscure. We will show data that indicate that TAL-1 and GATA-1, two transcrip-
176
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
tion factors essential for erythroid differentiation regulate the cell cycle during terminal erythroid differentiation. During the late phases of erythropoiesis, TAL-1 directly regulated the gene expression of two essential regulators of erythroid cell proliferation, Gfi-1b and the cyclin-dependent kinase inhibitor and G1 checkpoint regulator p21Cip. This transcriptional regulation is mediated by the association/dissociation of TAL-1 bound to the promoters of these two genes and ETO2, a known transcriptional repressor. We will also show that the differentiation factor GATA-1 directly interacted with the retinoblastoma (pRb) protein during the late stages of erythropoiesis. This association required the canonical LXCXE motif localized between amino acids 81 and 85 of GATA-1. This sequence is conserved during evolution, is not found in the other members of the GATA family and is absent in the short form of GATA-1 expressed in the acute megakaryoblastic leukemia of Down syndrome. The inhibition of cellular proliferation induced by GATA-1 over-expression was inefficient in pRb negative cell lines or when pRb was knocked down by specific shRNA. Using a GATA-1 protein mutated on the LXCXE motif (GATA-1Rb ), we showed that (i) the LXCXE motif was necessary for GATA-1/pRb association, (ii) the over-expression of GATA-1Rb did not inhibit cellular proliferation whereas GATA-1 over-expression did, and (iii) the expression of GATA-1Rb in a GATA-1-deficient erythroid cell line decreased the number of benzidine positive cells and increased the immature erythroid fraction. Finally, endogenous level expression of GATA-1Rb with GATA-1 HRD vector in the GATA-1.05 genotype mice resulted in a partial rescue of GATA-1.05 embryos from the early lethality at E11.5, but the embryos then exhibited severe anemia and died by E15.5. Analysis of the E12.5 fetal liver of these GATA-1Rb rescued mice showed an increased number of immature erythroid cells and a decreased number of mature erythroid cells. These results indicate that the GATA-1 and TAL-1 coordinate the expression of erythroid specific markers and the inhibition of cell division that occurs during the late stages of erythropoiesis. doi:10.1016/j.bcmd.2006.10.125
115 Activation of the LMO2 gene in human lymphoid cells by targeted insertion of a single retroviral LTR expression cassette into the first intron Byoung Y. Ryu 1, John T. Gray 1, David M. Bodine 2, Arthur W. Nienhuis 1 1 Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 2 Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD, USA The pathogenic activation of the LMO2 proto-oncogene contributed to leukemia in two children otherwise successfully treated for Severe Combined Immunodeficiency (SCID) in a gene therapy trial (Science 302:415–419, 2003). We have reproduced the retroviral vector insertion into the first intron of the LMO2
gene that occurred in one of the SCID patients in a human lympholeukemia cell line (Jurkat) using recombinant adenoassociated viral (rAAV) vector mediated gene targeting. The rAAV-LMO2 vector was constructed as follows. First, 1.3 kb fragments containing LMO2 intron sequences from upstream and downstream of the retroviral insertion site in the SCID patient were amplified by PCR from human genomic DNA and sequenced. Next, a gene cassette was designed to have the GFP coding sequences under the control of a single retroviral LTR that was used in the gene therapy trial followed by a polyadenylation signal, which in turn flanked by loxP sequences. This floxed LTR–GFP cassette was then placed in a reverse orientation between the 5V and 3V segments from LMO2 intron 1 and the fragments were cloned between AAV ITRs to derive the rAAV targeting vector. The Jurkat T cell line was transduced with rAAV–LMO2 particles and GFP expressing cells were selected by FACS at 9 days post-transduction and clones derived from single cells were recovered. The GFP-positive clones were screened for targeting into the LMO2 locus by PCR and Southern hybridization. Four clones showed the predicted bands in addition to wild-type bands indicating successful gene targeting into one of the LMO2 alleles. Next, LMO2 expression was compared to that in control Jurkat cells by real-time quantitative RT–PCR using the comparative CT method. Expression of LMO2 mRNA was at a very low level in untransduced Jurkat cells but was highly upregulated in all 4 clones ranging from an 1100- to 3500-fold increase. LMO2 protein was expressed in 4 clones but not in untransduced Jurkat consistent with real-time PCR results. Then, floxed DsRed expression cassette was constructed and used for Cre/loxP exchange in LMO2-targeted Jurkat clone. GFP expressing Jurkat cells were transduced with rAAV-Cre and transfected with DsRed cassette on following day. Single cell clones were isolated, expanded, and screened for DsRed expression without GFP expression. Southern hybridization was performed to confirm cassette exchange and four clones appeared to have cassette exchanged. Thus this system should prove useful for evaluating the relative safety of specific retroviral vector designs with respect to the potential for proto-oncogene activation. Currently, an exchange cassette containing globin LCR has been constructed and undergone Cre/loxP exchange. doi:10.1016/j.bcmd.2006.10.126
116 The expanding application of lentivirus-mediated erythroid-specific gene therapy Michel Sadelain, Selda Samakoglu, Alex Chang, Leszek Lisowski Memorial Sloan-Kettering Cancer Center, New York, NY, USA Recombinant lentiviruses are stable vectors that mediate efficient transfer of complex transcription units including large genomic fragments. We previously characterized in detail the vector termed TNS9, which encodes the human beta-globin gene, including an extended promoter both introns and both proximal enhancers, as well as large HS2, HS3 and HS4