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ABSTRACT: Locus control regions (LCRs) are transcriptional regulatory elements, which possess a dominant chromatin remodelling and transcriptional activating capability conferring full physiological levels of expression on a gene linked in cis, when integrated into the host cell genome. Using the human beta-globin LCR (betaLCR) as a model, we show that this class of control element can drive high levels of tissue-specific gene expression in stably transfected cultured cells from within an Epstein-Barr virus-based plasmid REV. Furthermore, a 38-kb betaLCR minilocus-REV cosmid vector was efficiently retained and maintained therapeutic levels of beta-globin transgene expression in the absence of drug selective pressure over a 2-month period of continuous culture equivalent to at least 60 generations. This demonstrates for the first time the feasibility of using REVs for gene therapy of the haemoglobinopathies. Importantly, our results demonstrate that as in the case of integrated transgenes, expression from within REVs is prone to silencing but that the inclusion of the betaLCR prevented this repression of gene function. Therefore, appropriate control elements to provide and maintain tissue-specific gene expression, as well as the episomal status of REVs is a crucial feature in vector design. Our data suggest that LCRs can contribute to this vital function.
Gene Therapy 04/2002; 9(5):327-36. · 3.71 Impact Factor
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ABSTRACT: We describe here for the first time the site of retention within the nucleus of pre-mRNA processing mutants unable to be exported to the cytoplasm. Fluorescence in situ hybridization was used to detect transcripts from human beta-globin genes that are either normal or defective in splicing or 3' end formation. Nuclear transcripts of both wild-type and mutant RNAs are detected only as intranuclear foci that colocalize with the template gene locus. The kinetics of transcript release from the site of transcription was assessed by treatment of cells with the transcriptional inhibitors actinomycin D, alpha-amanitin and DRB. These drugs induce the rapid disappearance of nuclear foci corresponding to wild-type human beta-globin RNA. In contrast, pre-mRNA mutants defective in either splicing or 3' end formation and which fail to be transported to the cytoplasm, are retained at the site of transcription. Therefore, 3' end processing and splicing appear to be rate limiting for release of mRNA from the site of transcription.
The EMBO Journal 06/1999; 18(10):2855-66. · 9.20 Impact Factor
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ABSTRACT: We describe the reproduction of the full pattern of expression of the muscle-specific desmin gene in transgenic mice using a 240-kb genomic clone spanning the human desmin locus. Analysis of RNA from adult tissues demonstrated that this fragment possesses all the necessary genetic regulatory elements required to provide reproducible, site-of-integration-independent, physiological levels of tissue-specific expression that is directly proportional to transgene copy number in all muscle cell types. In situ hybridization revealed that in marked contrast to murine desmin which is strongly expressed in the myotome of the somites, skeletal muscles, the heart, and smooth muscle of the vasculature by 9.5 days postcoitum, human desmin transgene expression was completely absent from smooth muscles, was very weak and restricted to the atrium and outflow tract within the heart, and was expressed at only 5% of murine desmin mRNA levels within the myotome of the somites. The spatial distribution and levels of human and mouse desmin expression were not coincident until 14.5 days postcoitum. Immunohistochemical analysis of human embryos at comparable stages of development showed that this transgene faithfully reproduces the human and not the mouse developmental expression pattern for this gene in transgenic mice. These results indicate that the 240-kb desmin genomic clone is capable of establishing an independent, chromatin domain in transgenic mice and provides the first definitive data for muscle-specific locus control region activity. In addition, our results demonstrate that the behavior of human transgenes in mice should, whenever possible, be compared to expression patterns for that gene in human embryonic as well as adult tissues.
Developmental Biology 10/1998; 201(1):26-42. · 4.07 Impact Factor
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ABSTRACT: The second intron (betaIVS-II) of the human beta-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantitative comparisons between betaIVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a beta-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3'-end formation and in the absence of exon skipping. Therefore, 3' cleavage of beta-globin transcripts requires the presence of betaIVS-II sequences, but not the splicing reaction. The placement of betaIVS-II in the IVS-I position did not reduce the efficiency of 3' cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of betaIVS-I in the intron II position, reduced the efficiency of 3'-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns betaIVS-I or alpha-globin IVS-II, only partially substitute (16 and 30% respectively) for betaIVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of betaIVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3'-end formation and may act as a post-transcriptional regulatory step in gene expression.
Nucleic Acids Research 03/1998; 26(3):721-9. · 8.03 Impact Factor
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ABSTRACT: The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain.
The EMBO Journal 02/1996; 15(2):319-33. · 9.20 Impact Factor
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ABSTRACT: DNA-protein interaction studies in vitro revealed several factors binding over the TATA box and the region of transcription initiation (cap) site of the human beta-globin promoter; TATA binding protein TBP at -30, Sp1 at -19, GATA-1 at -12 and +5, YY1 at -9 and a novel factor C1 over the site of initiation (-4 to +7). Point mutants which specifically abolish the binding of each of these proteins were tested in a beta-globin locus control region (LCR) construct which allows quantitative comparisons at physiological levels of transcription. Only mutants which drastically affect the binding of TBP resulted in decreased levels of transcription. A threshold value of TBP binding of 15-30% of wild type was sufficient to give normal levels of transcription. This indicates that the association of TF IID with the TATA box is not limiting in the rate of initiation of transcription.
Nucleic Acids Research 10/1995; 23(17):3473-80. · 8.03 Impact Factor
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ABSTRACT: Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.
Gene Therapy 08/1995; 2(5):336-43. · 3.71 Impact Factor
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ABSTRACT: Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.
Proceedings of the National Academy of Sciences 08/1995; 92(15):6728-32. · 9.68 Impact Factor
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ABSTRACT: In this paper we describe a complete deletional analysis of hypersensitive site three (HS3) of the human beta-globin Locus Control Region (LCR). The previously defined core fragment consists of 6 footprinted regions, with multiple binding sites for the erythroid-specific factor GATA-1 and G-rich motifs that can interact with ubiquitous factors such as Sp1 and TEF-2. We show in this paper that the 5' half of this fragment is the most important for activity in murine erythroleukemia (MEL) cells. A fragment containing footprints 1-4 can stimulate transcription of a linked human beta-globin gene to levels of about 40% of that obtained with footprints 1-6. Constructs containing either footprints 1-3 or 3-6 cannot be distinguished from the beta-globin gene alone. We further show that binding sites for the erythroid-specific factor NF-E2 can co-operatively interact with parts of the HS3 core fragment, and that HS3 requires elements upstream from -103 in the human beta-globin promoter for full activity. The importance of these results is discussed in the context of the regulation of the genes in the human beta-globin cluster.
Biochimica et Biophysica Acta 11/1994; 1219(2):351-60. · 4.66 Impact Factor
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ABSTRACT: Murine erythroleukemia (MEL) cells are erythroid progenitors that can be induced to undergo terminal erythroid differentiation in culture. We have used MEL cells here as a model system to study the nuclear organization of splicing snRNPs during the physiological changes in gene expression which accompany differentiation. In uninduced MEL cells, snRNPs are widely distributed throughout the nucleoplasm and show an elevated concentration in coiled bodies. Within the first two days after induction of terminal erythroid differentiation, the pattern of gene expression changes, erythroid-specific transcription is activated and transcription of many other genes is repressed. During this early stage splicing snRNPs remain widely distributed through the nucleoplasm and continue to associate with coiled bodies. At later stages of differentiation (four to six days), when total transcription levels have greatly decreased, splicing snRNPs are redistributed. By six days postinduction snRNPs were concentrated in large clusters of interchromatin granules and no longer associated with coiled bodies. At the end-point of erythroid differentiation, just before enucleation, we observe a dramatic segregation of splicing snRNPs from the condensed chromatin. Analysis by EM shows that the snRNPs are packaged into a membrane-associated structure at the nuclear periphery which we term the "SCIM" domain (i.e., SnRNP Clusters Inside a Membrane).
The Journal of Cell Biology 01/1994; 123(5):1055-68. · 10.26 Impact Factor
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ABSTRACT: This paper describes the mechanism of regulation of the human beta-globin on the basis of a number of natural mutations and experiments in transgenic mice. From these data we conclude that this multigene locus is regulated at a number of different levels involving specific interactions between the Locus Control Region (LCR) and the individual genes. Most important is the action of stage specific transcription factors acting on sequences immediately flanking the genes. In addition, specificity is obtained through specific interaction of the genes with the LCR and through competition of the genes for interaction with the LCR.
Philosophical Transactions of The Royal Society B Biological Sciences 03/1993; 339(1288):183-91. · 6.40 Impact Factor
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ABSTRACT: Fundamental studies of the mechanism of human beta-like globin gene-expression have identified DNA sequences (locus control regions or LCRs) which directly influence the specific activation of beta-globin genes in erythroid cells. Here we report the first applications of LCR-mediated gene-activation to stable electrophysiological expression of several homomultimeric ion channel proteins. We describe expression driven from a native K+ channel gene promoter region, contiguous to an intronless coding sequence, within a single excised human genomic DNA fragment. In addition, cDNAs encoding both inactivating and non-inactivating mammalian K+ currents have been expressed by insertion between the promoter and second intron of the human beta-globin gene. Expression levels are characteristically independent of integration position and are proportional to LCR-gene copy number, a parameter specific for each clonal cell line. K+ channel expression is inducible by erythroid differentiation and has been demonstrated by electrophysiological recordings of cells taken directly from culture.
Receptors and Channels 02/1993; 1(1):25-37.
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Cold Spring Harbor Symposia on Quantitative Biology 02/1993; 58:7-13.
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ABSTRACT: We have tested the effect of the individual DNase I hypersensitive site (HS) regions of the globin locus control region (LCR) on the developmental expression pattern of the human gamma and beta genes in transgenic mice. The results show that HS3 is the most active site during the embryonic period. It is also the only site capable of high level expression of the gamma genes during fetal hematopoiesis, in a population of cells that are capable of expressing both the gamma and beta genes. Region HS4 shows the highest activity during the adult stage and expresses the gamma genes only at low levels during the embryonic period. HS2 drives equivalent levels of gamma or beta transgene expression throughout development. HS1 has a similar pattern to HS2, although the activity of HS1 is very low. From these results we conclude that the HS regions have distinct developmental specificities and suggest that in the complete LCR they interact with each other to form a larger complex which, in turn, interacts with the globin genes.
Genes & Development 02/1993; 7(1):106-13. · 11.66 Impact Factor
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• N. Dillon, M Antoniou,
M.Berry,
E. DeBoer,
D. Drabek,
J.Ellis,
P. Fraser,
O. Hanscombe,
A.Imam,
M-A. Koken,
M. Lindenbaum,
D. Meijer,
S. Philipsen,
S. Pruzina,
S. Raguz-Bolognesi,
J. Strouboulis,
D. Talbot,
D. Whyatt,
F. Grosveld
01/1993: pages pp. 149-159;
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ABSTRACT: We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.
Nucleic Acids Research 04/1992; 20(5):997-1003. · 8.03 Impact Factor
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ABSTRACT: We have studied the interaction between the dominant control region (DCR) and the promoter of the human beta-globin gene. Expression analysis in MEL cells has revealed that the DCR contains a number of elements capable of replacing the upstream (-250 to -100) erythroid-specific region of the promoter. The DCR strongly stimulates expression from a promoter possessing only a TATA box. However, this basic level of transcription is not induced upon erythroid differentiation of the cells. Mutational analysis of the minimal (-100, noninducible) promoter shows that only the combination of the DCR and the CAC/CCAAT elements provides erythroid-specific transcription. These regions act synergistically to produce full regulated expression during erythroid differentiation.
Genes & Development 07/1990; 4(6):1007-13. · 11.66 Impact Factor
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ABSTRACT: The human beta-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human beta-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron.
The EMBO Journal 02/1990; 9(1):233-40. · 9.20 Impact Factor
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ABSTRACT: The regulatory elements that determine the expression pattern of a number of eukaryotic genes expressed specifically in certain tissues have been defined and studied in detail. In general, however, the expression conferred by these elements on genes reintroduced into the genomes of cell lines and transgenic animals has turned out to be at a low level relative to that of endogenous genes, and influenced by the chromosomal site of insertion of the exogenous construct. We have previously shown that if regions flanking the human beta-globin locus are introduced into the mouse genome along with the human beta-globin gene, a level of expression comparable to that of endogenous genes can be achieved that is also independent of integration site. We have now defined a dominant control region with these properties consisting of 6.5 kilobases of DNA encompassing erythroid cell-specific DNase I hypersensitive sites. The identification of such dominant control regions could have important applications in somatic gene therapy.
Nature 04/1989; 338(6213):352-5. · 36.28 Impact Factor
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Progress in clinical and biological research 02/1989; 316A:1-13.
