- Email: [email protected]
Long-range function of the ERV-9 LTR in transcriptional regulation of the globin genes
182
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
are essential for high-level expression in erythroid cells. In this paper we demonstrate by chromatin immunoprecipitation (ChIP) that Erythroid Krupple-like Factor (EKLF) binds to embryonic/fetal globin gene promoters in primitive but not in definitive erythroid cells. EKLF binds strongly to adult globin gene promoters and to LCR sequences HS4, HS3, HS2 and HS1 in both primitive and definitive erythroid cells. Trimethylation of histone H3K4 and H3K27 at the embryonic/fetal and adult globin gene promoters is equivalent in definitive cells; therefore, the differential binding of EKLF to these promoters does not appear to result from changes in chromatin configuration. Interestingly, the level of EKLF in definitive cells is threefold higher than the level in primitive cells. These results suggest that temporalspecific changes in EKLF abundance result in differential binding of this essential, erythroid transcription factor to embryonic/fetal globin gene promoters during development and that these changes in EKLF binding specificity mediate the competitive interactions of globin gene family members with the LCR. doi:10.1016/j.bcmd.2006.10.139
129 Cellular and myeloablative requirements for correction of murine SCD Marie Trudel, Hady Felfly Institut de recherches cliniques de Montreal, Universite de Montreal, Montreal, Quebec, Canada Permanent correction of sickle cell disease (SCD) requires the establishment of basic criteria that could serve as clinical reference. Our previous findings have indicated that bone marrow transplantation of normal donor cells into a sickle cell recipient would have a strong selective advantage and predominate upon erythroid differentiation, maturation and survival. Using our SAD mouse model of SCD on a homogenous genetic background, we determined the minimal number of normal bone marrow cells necessary to correct the disease. We generated by Competitive Repopulating Assay 12 chimeric groups of SAD mice with at least 5 mice per group, ranging from 0 to 100% normal white blood cell (WBC) chimerism. Analysis of 150 SAD recipient mice for short and long-term post-transplantation (2 months to > 1 year) was carried out. These analyses showed that the group with ¨ 21% to 26% normal WBC chimerism in SAD mice, determined by the Gpi1a marker, produced, respectively ¨ 40% to 50% of normal RBCs in peripheral blood. With 21% to 26% normal cell chimerism in SAD mice, RBC parameters were significantly improved and their half-life was increased by ¨ 30%. These improvements lead to increased erythropoiesis efficiency in bone marrow and spleen. Consequently, pathological analysis of chimeric SAD mice showed reduced splenic RBC sequestration, hematopoiesis/erythropoiesis and significantly decreased splenomegaly. Moreover, the life span of the group with 26% chimerism in SAD mice reached normal life
expectancy. Together, our results established a range of 21% to 26% of normal bone marrow cells as appropriate for a significant and long-term physiologic correction of SAD mouse condition in vivo. Based on this result, we then aimed to determine the most efficient conditions of bone marrow transplantation to achieve the same therapeutic range but with minimal myeloablation. This was addressed with a series of transplantations at 100 or 200 rad of irradiation and, for each of them, the optimal number of cells and time of cell transfer were assessed. Transplantation experiments in SAD recipients at 200 rad comparing transfer of 3 bone marrow cell doses (20 – 50 million total cells) and time of cell transfer (4 or 52 h) showed that SAD mice chimerism were well above the therapeutic range (37 – 76%). Transplantation experiments in SAD recipients at 100 rad with transfer of 3 bone marrow cell doses (40 – 80 million total cells) and time of cell transfer (4 or 28 h) displayed SAD chimerism within the therapeutic range (21 –59%). In both series of transplantations, similar proportion of mice engrafted and chimerism seem to improve with increasing number of cells. In summary, we established the minimal criteria for cellular correction of SCD at 21– 26% normal cells and demonstrated appropriate chimerism efficiency with minimal myeloablative condition, which could have direct implications for the design of cellular/gene therapy approaches in human SCD. doi:10.1016/j.bcmd.2006.10.140
130 Long-range function of the ERV-9 LTR in transcriptional regulation of the globin genes Dorothy Tuan, Wenhu Pi, Xingguo Zhu, Min Wu, Yongchao Wang, Jianhua Ling Department of Biochemistry and Mol. Biology, Medical College of Georgia, Augusta, GA, USA The solitary LTRs of the ERV-9 human endogenous retrovirus are present at 3000 –4000 copies in the human genome. They are associated with a number of hematopoietic genes. In the h-globin gene locus, a solitary ERV-9 LTR is juxtaposed to the HS5 site in the 5V boundary of the h-globin Locus Control Region (LCR) at a distance 25, 40 and 70 kb from the (-, g- and h-globin genes respectively. The ERV-9 LTR contains the U3 region spanning the retroviral enhancer and promoter, the transcribed R and U5 regions, but no internal gag, pol and env genes. The U3 enhancer spans 14 tandem repeats of 40 bases with recurrent CCAAT, GATA and GTGGGGA motifs that bind, respectively, the ubiquitous transcription factor NF-Y and the hematopoietic factors GATA-2 and MZF-1 to assemble an active enhancer complex NF-Y/GATA-2/MZF1 in K562 cells. In transfection assays and in transgenic zebrafish, the ERV-9 LTR exhibits prominent enhancer activity in progenitor cells including erythroid progenitor cells. To study its function in transcriptional regulation of the far downstream globin genes, we have generated two independent lines of LTR transgenic (Tg) mice
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
carrying a 100 kb BAC DNA spanning the entire human hglobin gene locus. From these lines of Tg mice we deleted the ERV-9 LTR by using Cre-loxP mediated in situ recombination to generate the two derivative lines of DLTR Tg mice. We analyzed the transcription patterns of the human globin transgenes in fetal livers of day 12.5 and 16.5 embryos and in bone marrows and spleen erythroid cells of adult Tg mice. The results show that the g- to h-globin gene switching normally taking place in the LTR Tg mice is impaired in the DLTR Tg mice. The mechanism by which the ERV-9 LTR regulates g- and h-globin gene switching over the long distance is under investigation. doi:10.1016/j.bcmd.2006.10.141
131 Interplay between DNA and histone methylation at an A-globin gene silenced by antisense RNA M. De Gobbi 1, J.A. Sloane Stanley 1, H. Ayyub, G.W. Wood 1, D.R. Higgs 1, C. Tufarelli 2 1 MRC Molecular Haematology Unit, WIMM Oxford OX3 9DS, UK 2 Department of Genetics, University of Leicester, Leicester LE1 7RH, UK We have previously shown that in a patient with athalassaemia, in a transgenic mouse model and in differentiating mouse ES cells, antisense-RNA transcripts running through the a-globin CpG island lead to its full methylation. These RNAs act exclusively in cis as they are unable to trigger methylation of a trans allele, both in the patient and in transgenic mice. We have now analysed the histone modifications associated with the silenced and methylated locus in the patient and control cell lines, and in differentiating mouse ES cells. We found that the chromatin of the silenced and methylated aglobin gene is enriched in histone H3 trimethylated at residue K9. This modification is unique to the methylated allele and is never found in cell lines from non-erythroid tissues of normal individuals. Interestingly, as seen with DNA methylation, H3K9 trimethylation is very localised and restricted to the CpG island of the silenced a-globin gene. The analysis of ES cells at different time points during differentiation indicates that in this system the establishment of DNA and histone methylation is concurrent with a major peak between day 3 and day 4 of differentiation. These findings indicate that, consistent with what is seen in other systems, RNA mediated chromatin silencing is associated with trimethylated H3K9 and suggests that at least in this system the enzymatic activities responsible for RNA-directed DNA methylation and H3K9 trimethylation may be the component of the same complex. doi:10.1016/j.bcmd.2006.10.142
183
132 Lentiviral vector non-coding elements that impart high titers and expression to B-globin cassettes Fabrizia Urbinati, Ping Xia, Anil Perumbeti, Ming Xia, Andrea Corsinotti, Alex Zarzuela, Natalya Perelman, Punam Malik Childrens Hospital Los Angeles, Los Angeles, CA, USA Thalassemias are the most common monogenic defects that result from absent/reduced h-globin in red blood cells (RBC). The introduction of a functional b-globin gene (hh) in hematopoietic stem cells (HSCs) could permanently correct the defect and normalize survival of RBCs derived from modified HSCs. However, the complexities of the hh and its regulatory elements have been significant obstacles for optimization of gene therapy approaches using g-oncoretroviral vectors. Recently, lentiviral vectors were shown to enable stable and efficient transmission of the hh and their regulatory elements that were previously found to be unstable in oncoretroviral vectors, result in high titer virus and therapeutic levels of expression. Our goal was to identify non-coding elements in lentiviral vectors required for this effect. A series of lentiviral vectors carrying different cis-elements from the gag and env fragments were made. All vectors encoded human hb gene driven by h-regulatory elements (h-promoter, HS2, HS3, HS4 and h3V enhancer). All vectors were packaged in 293T cells, and assayed in differentiated mouse erythroleukemia (MEL) cells. The provirus was stably transmitted from all the vectors, including one of the completely Fgutted vectors. Titration of these vectors showed that the rev/RRE in HIV is likely the most important element imparting high titers to the vector, followed by the Env splice acceptor and Gag. The hh expression was analyzed both at the RNA and at the protein level (FACS analysis). The mean fluorescence intensity of hh expression in differentiated MEL cells was similar with all vectors, suggesting that upon integration, viral cis elements probably play a minimal role in improving expression of hh transcripts, although this is still under investigation. Data will be presented showing the role of the various lentiviral vectors cis-elements contributing in high titer vector production, high expression and stability of hh gene. doi:10.1016/j.bcmd.2006.10.143
133 The barrier activity of the chicken hypersensitive site-4 insulator (cHS4) element result in higher human beta-globin expression from lentiviral vectors Paritha I. Arumugam, Fabrizia Urbinati, Jessica Scholes, Ping Xia, Natalya Perelman, Alex Zarzuela, Jiing-Kuan Yee, Punam Malik Childrens Hospital Los Angeles, Los Angeles, CA, USA Lentiviral vectors (LV) carrying the human h-globin gene (hh) and locus control region (LCR) have changed the field
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
are essential for high-level expression in erythroid cells. In this paper we demonstrate by chromatin immunoprecipitation (ChIP) that Erythroid Krupple-like Factor (EKLF) binds to embryonic/fetal globin gene promoters in primitive but not in definitive erythroid cells. EKLF binds strongly to adult globin gene promoters and to LCR sequences HS4, HS3, HS2 and HS1 in both primitive and definitive erythroid cells. Trimethylation of histone H3K4 and H3K27 at the embryonic/fetal and adult globin gene promoters is equivalent in definitive cells; therefore, the differential binding of EKLF to these promoters does not appear to result from changes in chromatin configuration. Interestingly, the level of EKLF in definitive cells is threefold higher than the level in primitive cells. These results suggest that temporalspecific changes in EKLF abundance result in differential binding of this essential, erythroid transcription factor to embryonic/fetal globin gene promoters during development and that these changes in EKLF binding specificity mediate the competitive interactions of globin gene family members with the LCR. doi:10.1016/j.bcmd.2006.10.139
129 Cellular and myeloablative requirements for correction of murine SCD Marie Trudel, Hady Felfly Institut de recherches cliniques de Montreal, Universite de Montreal, Montreal, Quebec, Canada Permanent correction of sickle cell disease (SCD) requires the establishment of basic criteria that could serve as clinical reference. Our previous findings have indicated that bone marrow transplantation of normal donor cells into a sickle cell recipient would have a strong selective advantage and predominate upon erythroid differentiation, maturation and survival. Using our SAD mouse model of SCD on a homogenous genetic background, we determined the minimal number of normal bone marrow cells necessary to correct the disease. We generated by Competitive Repopulating Assay 12 chimeric groups of SAD mice with at least 5 mice per group, ranging from 0 to 100% normal white blood cell (WBC) chimerism. Analysis of 150 SAD recipient mice for short and long-term post-transplantation (2 months to > 1 year) was carried out. These analyses showed that the group with ¨ 21% to 26% normal WBC chimerism in SAD mice, determined by the Gpi1a marker, produced, respectively ¨ 40% to 50% of normal RBCs in peripheral blood. With 21% to 26% normal cell chimerism in SAD mice, RBC parameters were significantly improved and their half-life was increased by ¨ 30%. These improvements lead to increased erythropoiesis efficiency in bone marrow and spleen. Consequently, pathological analysis of chimeric SAD mice showed reduced splenic RBC sequestration, hematopoiesis/erythropoiesis and significantly decreased splenomegaly. Moreover, the life span of the group with 26% chimerism in SAD mice reached normal life
expectancy. Together, our results established a range of 21% to 26% of normal bone marrow cells as appropriate for a significant and long-term physiologic correction of SAD mouse condition in vivo. Based on this result, we then aimed to determine the most efficient conditions of bone marrow transplantation to achieve the same therapeutic range but with minimal myeloablation. This was addressed with a series of transplantations at 100 or 200 rad of irradiation and, for each of them, the optimal number of cells and time of cell transfer were assessed. Transplantation experiments in SAD recipients at 200 rad comparing transfer of 3 bone marrow cell doses (20 – 50 million total cells) and time of cell transfer (4 or 52 h) showed that SAD mice chimerism were well above the therapeutic range (37 – 76%). Transplantation experiments in SAD recipients at 100 rad with transfer of 3 bone marrow cell doses (40 – 80 million total cells) and time of cell transfer (4 or 28 h) displayed SAD chimerism within the therapeutic range (21 –59%). In both series of transplantations, similar proportion of mice engrafted and chimerism seem to improve with increasing number of cells. In summary, we established the minimal criteria for cellular correction of SCD at 21– 26% normal cells and demonstrated appropriate chimerism efficiency with minimal myeloablative condition, which could have direct implications for the design of cellular/gene therapy approaches in human SCD. doi:10.1016/j.bcmd.2006.10.140
130 Long-range function of the ERV-9 LTR in transcriptional regulation of the globin genes Dorothy Tuan, Wenhu Pi, Xingguo Zhu, Min Wu, Yongchao Wang, Jianhua Ling Department of Biochemistry and Mol. Biology, Medical College of Georgia, Augusta, GA, USA The solitary LTRs of the ERV-9 human endogenous retrovirus are present at 3000 –4000 copies in the human genome. They are associated with a number of hematopoietic genes. In the h-globin gene locus, a solitary ERV-9 LTR is juxtaposed to the HS5 site in the 5V boundary of the h-globin Locus Control Region (LCR) at a distance 25, 40 and 70 kb from the (-, g- and h-globin genes respectively. The ERV-9 LTR contains the U3 region spanning the retroviral enhancer and promoter, the transcribed R and U5 regions, but no internal gag, pol and env genes. The U3 enhancer spans 14 tandem repeats of 40 bases with recurrent CCAAT, GATA and GTGGGGA motifs that bind, respectively, the ubiquitous transcription factor NF-Y and the hematopoietic factors GATA-2 and MZF-1 to assemble an active enhancer complex NF-Y/GATA-2/MZF1 in K562 cells. In transfection assays and in transgenic zebrafish, the ERV-9 LTR exhibits prominent enhancer activity in progenitor cells including erythroid progenitor cells. To study its function in transcriptional regulation of the far downstream globin genes, we have generated two independent lines of LTR transgenic (Tg) mice
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
carrying a 100 kb BAC DNA spanning the entire human hglobin gene locus. From these lines of Tg mice we deleted the ERV-9 LTR by using Cre-loxP mediated in situ recombination to generate the two derivative lines of DLTR Tg mice. We analyzed the transcription patterns of the human globin transgenes in fetal livers of day 12.5 and 16.5 embryos and in bone marrows and spleen erythroid cells of adult Tg mice. The results show that the g- to h-globin gene switching normally taking place in the LTR Tg mice is impaired in the DLTR Tg mice. The mechanism by which the ERV-9 LTR regulates g- and h-globin gene switching over the long distance is under investigation. doi:10.1016/j.bcmd.2006.10.141
131 Interplay between DNA and histone methylation at an A-globin gene silenced by antisense RNA M. De Gobbi 1, J.A. Sloane Stanley 1, H. Ayyub, G.W. Wood 1, D.R. Higgs 1, C. Tufarelli 2 1 MRC Molecular Haematology Unit, WIMM Oxford OX3 9DS, UK 2 Department of Genetics, University of Leicester, Leicester LE1 7RH, UK We have previously shown that in a patient with athalassaemia, in a transgenic mouse model and in differentiating mouse ES cells, antisense-RNA transcripts running through the a-globin CpG island lead to its full methylation. These RNAs act exclusively in cis as they are unable to trigger methylation of a trans allele, both in the patient and in transgenic mice. We have now analysed the histone modifications associated with the silenced and methylated locus in the patient and control cell lines, and in differentiating mouse ES cells. We found that the chromatin of the silenced and methylated aglobin gene is enriched in histone H3 trimethylated at residue K9. This modification is unique to the methylated allele and is never found in cell lines from non-erythroid tissues of normal individuals. Interestingly, as seen with DNA methylation, H3K9 trimethylation is very localised and restricted to the CpG island of the silenced a-globin gene. The analysis of ES cells at different time points during differentiation indicates that in this system the establishment of DNA and histone methylation is concurrent with a major peak between day 3 and day 4 of differentiation. These findings indicate that, consistent with what is seen in other systems, RNA mediated chromatin silencing is associated with trimethylated H3K9 and suggests that at least in this system the enzymatic activities responsible for RNA-directed DNA methylation and H3K9 trimethylation may be the component of the same complex. doi:10.1016/j.bcmd.2006.10.142
183
132 Lentiviral vector non-coding elements that impart high titers and expression to B-globin cassettes Fabrizia Urbinati, Ping Xia, Anil Perumbeti, Ming Xia, Andrea Corsinotti, Alex Zarzuela, Natalya Perelman, Punam Malik Childrens Hospital Los Angeles, Los Angeles, CA, USA Thalassemias are the most common monogenic defects that result from absent/reduced h-globin in red blood cells (RBC). The introduction of a functional b-globin gene (hh) in hematopoietic stem cells (HSCs) could permanently correct the defect and normalize survival of RBCs derived from modified HSCs. However, the complexities of the hh and its regulatory elements have been significant obstacles for optimization of gene therapy approaches using g-oncoretroviral vectors. Recently, lentiviral vectors were shown to enable stable and efficient transmission of the hh and their regulatory elements that were previously found to be unstable in oncoretroviral vectors, result in high titer virus and therapeutic levels of expression. Our goal was to identify non-coding elements in lentiviral vectors required for this effect. A series of lentiviral vectors carrying different cis-elements from the gag and env fragments were made. All vectors encoded human hb gene driven by h-regulatory elements (h-promoter, HS2, HS3, HS4 and h3V enhancer). All vectors were packaged in 293T cells, and assayed in differentiated mouse erythroleukemia (MEL) cells. The provirus was stably transmitted from all the vectors, including one of the completely Fgutted vectors. Titration of these vectors showed that the rev/RRE in HIV is likely the most important element imparting high titers to the vector, followed by the Env splice acceptor and Gag. The hh expression was analyzed both at the RNA and at the protein level (FACS analysis). The mean fluorescence intensity of hh expression in differentiated MEL cells was similar with all vectors, suggesting that upon integration, viral cis elements probably play a minimal role in improving expression of hh transcripts, although this is still under investigation. Data will be presented showing the role of the various lentiviral vectors cis-elements contributing in high titer vector production, high expression and stability of hh gene. doi:10.1016/j.bcmd.2006.10.143
133 The barrier activity of the chicken hypersensitive site-4 insulator (cHS4) element result in higher human beta-globin expression from lentiviral vectors Paritha I. Arumugam, Fabrizia Urbinati, Jessica Scholes, Ping Xia, Natalya Perelman, Alex Zarzuela, Jiing-Kuan Yee, Punam Malik Childrens Hospital Los Angeles, Los Angeles, CA, USA Lentiviral vectors (LV) carrying the human h-globin gene (hh) and locus control region (LCR) have changed the field