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PubMed2004

An Erythroid-Specific Chromatin Opening Element Increases β-Globin Gene Expression from Integrated Retroviral Gene Transfer Vectors.

Gene therapy strategies requiring long-term high-level expression from integrated genes are currently limited by inconsistent levels of expression. This may be observed as variegated, silenced or position-dependent gene expression. Each of these phenomena involve suppressive chromatin structures. We hypothesized that by actively conferring an open chromatin structure on integrated vectors would increase transgene expression. To test this idea we used a 100bp element from the β-globin locus control region (LCR) which is able to independently open local chromatin structure in erythroid tissues. This element includes binding sites for GATA-1, NF-E2, EKLF and Sp-1 and is evolutionarily conserved. We constructed a series of MSCV-based vectors containing the β-globin gene driven by a minimal β-globin promoter with combinations of the HSFE and LCR derived enhancer elements. Pools of MEL clones containing integrated vectors were analyzed for chromatin structure and β-globin gene expression. The HSFE increased the extent of nuclease sensitive chromatin over the promoters of the constructs. The most effective vector included tandem copies of the HSFE and produced a 5-fold increase in expression compared to the promoter alone. These results indicate that the HSFE is able to augment the opening of β-globin promoter chromatin structure and significantly increase gene expression in the context of an integrated retroviral vector.

Journal Article↗
PubMed1991

[Current trends in hemoglobin genetic diseases].

The current aspects of genetic diseases of hemoglobin are as follows. (1) The localisation and the dimension of at-risk populations for sickle cell disease and thalassemia in metropolitan France are not exactly known. However, some recent epidemiological studies and preliminary results of studies being currently underway allow to delimitate the problem. The at-risk populations are localized in the parisian area, in Provence-Côte d'Azur, in the Lyon and Grenoble areas, in Lorraine, Alsace and the Northern part of France. The number of heterozygotes for these diseases is situated between 250,000 and 350,000. The number of homozygotes born each year is between 100 and 150. More than 250 homozygous thalassemia patients and probably 2,000 to 3,000 sickle cell patients are living at this time in metropolitan France. (2) The transgenic mouse model is an experimental tool which allowed important advances in the knowledge of the hemoglobin human genome. Thus, we now have lines of mice expressing human fetal (g genes) and human adult (b genes) hemoglobins. Recently, enhancer sequences have been identified in 5' to the embryonic gene epsilon. When this region, named locus-region-control (LCR), is inserted along with g and b globin genes, their expression is observed at a very high level. The main objective of this technique is to obtain mouse model for sickle cell disease and thalassemia for studies on the pathophysiology and the therapy of these diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Sickle Cell↗
PubMed1991

Regulation of human fetal and adult globin genes in mouse erythroleukemia cells.

We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta-globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5' end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.

Adenine Phosphoribosyltransferase↗
PubMed1994

Fetal hemoglobin induction by acetate, a product of butyrate catabolism.

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma-chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF-containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two-carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

Acetates↗
PubMed1995

Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer.

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.

Animals↗
PubMed1994

Anaemia and resistance to malaria in transgenic mice expressing human tumour necrosis factor.

Transgenic mice carrying a modified human tumour necrosis factor (huTNF)/beta-globin gene construct linked to the T-cell-specific locus control region of the human CD2 gene express huTNF in their T cells which is released into the circulation and causes the development of a wasting syndrome. We now report that the mice develop anaemia, probably through enhanced erythrophagocytosis rather than inhibition of reticulocyte production. Thus autologous erythrocytes, as well as sheep erythrocytes, were cleared more rapidly from the circulation of transgenic mice than from littermate controls. By contrast, peritoneal macrophages from transgenic mice were less phagocytic in vitro than cells from controls. They also secreted less murine (mu)TNF when stimulated by either bacterial lipopolysaccharide or toxic malarial antigens. The yields of muTNF approached normal levels, however, when these refractory cells from the transgenic mice were stimulated in the presence of a high concentration of indomethacin, suggesting that the production of muTNF was inhibited by enhanced synthesis of prostaglandins. The parasitaemia of transgenic mice infected with Plasmodium yoelii was about 10-fold less at its peak than in controls, although it followed the same time-course, and the multiplication of P. chabaudi was inhibited to an even greater degree. This control of parasitaemia may also be explained by enhancement of macrophage activity, mediated by huTNF acting on the murine p55 receptor, presumably by increasing the removal of parasites by phagocytosis or their killing by toxic products released by the activated macrophages. These observations suggest that a factor in the anaemia of human malaria may be macrophage activation caused by the secretion of TNF that occurs in this disease.

Anemia↗
PubMed1994

The leucine zipper is necessary for stabilizing a dimer of the helix-loop-helix transcription factor USF but not for maintenance of an elongated conformation.

The basic helix-loop-helix transcription factor USF43 binds to E box motifs on certain promoters and enhancers, as well as the beta-globin locus control region. We have used gel filtration chromatography, velocity centrifugation, and chemical cross-linking methods to investigate the stoichiometry and shape of USF43 in solution and when bound to DNA. USF43 has a very large Stokes' radius (44 A) and a high frictional ratio (1.64), consistent with an asymmetric elongated oligomer. Under a variety of conditions, the only detectable USF43 species in solution and bound to DNA is a dimer. The carboxyl-terminal leucine zipper is absolutely essential for a stable dimer but not for the elongated conformation. We used a protease footprinting assay to demonstrate that, when USF43 binds to DNA, a approximately 15-kDa USF43 domain becomes resistant to cleavage with trypsin. This domain includes sequences that are not expected to interact with the DNA helix, suggesting that trypsin cleavage sites are masked by a conformational change. Our results show that the oligomerization state of USF43 does not change upon binding to DNA, and the helix-loop-helix oligomerization motif of USF43 is not itself sufficient to form a high affinity dimerization interface.

Amino Acid Sequence↗
PubMed1993

Regulated expression of K+ channel genes in electrically silent mammalian cells by linkage to beta-globin gene-activation elements.

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.

Amino Acid Sequence↗
PubMed1993

Genetic heterogeneity among blue-cone monochromats.

Thirty-three unrelated subjects with blue-cone monochromacy or closely related variants of blue-cone monochromacy were examined for rearrangements in the tandem array of genes encoding the red- and green-cone pigments. In 24 subjects, eight genotypes were found that would be predicted to eliminate the function of all of the genes within the array. As observed in an earlier study, the rearrangements involve either deletion of a locus control region adjacent to the gene array or loss of function via homologous recombination and point mutation. One inactivating mutation, Cys203-to-Arg, was found in 15 probands who carry single genes and in both visual pigment genes in one subject whose array has two genes. This mutation was also found in at least one of the visual pigment genes in 1 subject whose array has multiple genes and in 2 of 321 control subjects, suggesting that preexisting Cys203-to-Arg mutations constitute a reservoir of chromosomes that are predisposed to generate blue-cone-monochromat genotypes by unequal homologous recombination and/or gene conversion. Two other point mutations were identified: (a) Arg247-to-Ter in one subject with a single red-pigment gene and (b) Pro307-to-Leu in one subject with a single 5' red-3' green hybrid gene. The observed heterogeneity of genotypes points to the existence of multiple one- and two-step mutational pathways to blue-cone monochromacy.

Base Sequence↗
PubMed1993

Analysis of the human zeta-globin gene promoter in transgenic mice.

zeta-Globin is the embryonic form of the alpha chain of hemoglobin. Transgenic mice generated with zeta-globin constructs containing the zeta-globin gene, 557 bp of 5' flanking sequence, and 2-kb of 3' flanking sequence linked to the beta-globin locus control region hypersensitive site 2 (HS2) expressed human zeta-globin only in embryonic yolk sac erythroid tissue, and not in definitive erythroid tissue in the fetal liver or in adult peripheral blood. To determine what sequences in the 5' flanking region of the zeta-globin gene might be important for developmental specificity, a series of 5' deletion constructs of the zeta-globin gene were made and used to generate transgenic mice. The 5' ends of these constructs were located 417, 207, and 128 bp 5' to the zeta-globin transcriptional start site, and HS2 was included to increase the level of erythroid-specific expression. In all lines of mice tested, human zeta-globin was expressed only in embryonic tissue, and not in fetal livers or in adult peripheral blood. Expression was independent of copy number and appeared to be dependent on the site of transgene insertion. These data suggest that the proximal 128 bp of the zeta-globin promoter is sufficient to properly regulate zeta-globin expression during development.

Animals↗
PubMed1993

Wasting, ischemia, and lymphoid abnormalities in mice expressing T cell-targeted human tumor necrosis factor transgenes.

To evaluate the biologic potential of T cell-specific TNF production in vivo, we have generated transgenic mice constitutively expressing TNF in their T cell compartment. This was achieved by placing a wild-type or a 3'-UTR modified fragment of the human TNF gene under the influence of the T cell-specific, locus control region of the human CD2 gene. Transgenic mice that express human TNF mRNA in T cells develop marked histologic and cellular changes locally in their lymphoid organs and a lethal wasting syndrome associated with widespread vascular thrombosis and tissue necrosis. The extent of pathologic changes and their time of onset appear to reflect levels of transgene expression. Thus, transgenic lines that express the transgene at high levels show both lymphoid organ and systemic abnormalities with wasting. In one transgenic line, mice express lower levels of the transgene and develop normally despite pronounced local lymphoid organ defects, confirming in vivo, the differential potential of localized and systemic TNF action. All pathologic changes could be neutralized by the administration of mAb specific for human TNF. These results demonstrate the important role of T cell-specific TNF production in the development of specific pathology and provide a means by which to evaluate the role of TNF in thymocyte development. Transgenic mice that express TNF constitutively in the T cell compartment offer a unique in vivo system by which to analyze the molecular character of systemic vs contact-dependent and paracrine modes of TNF action. Furthermore, given the species-specific nature of the mouse p75 TNF receptor, it is assumed that the pathology induced by human TNF in these transgenic mice is associated exclusively with p55 TNF receptor signaling. Conceivably, the differential contribution of each of the two TNF receptors in thymus development and TNF-mediated disease can be assessed by comparison of the biologic potential of human vs mouse TNF in the transgenic system developed.

Animals↗
PubMed1993

The major regulatory element upstream of the alpha-globin gene has classical and inducible enhancer activity.

A major positive regulatory element has recently been identified 40 kb upstream from the human zeta 2-globin gene. This regulatory element increases the expression of a linked alpha-globin gene in mouse erythroleukemia cells and in transgenic mice. This element has been shown to share many of the structural and functional features of the locus control region (LCR) of the beta-globin gene cluster. We have examined the activity of a small fragment from this regulatory domain (alpha LCR) in a transient expression system. We show that this element is active as an enhancer in the erythroid environment of K562 cells. It is somewhat less effective as an enhancer in the nonerythroid environment of HeLa cells. This alpha LCR fragment does not exhibit promoter specificity because it can activate both the promoter of its endogenous target gene and the heterologous promoter of the SV40 early genes. Although the major activity of this element is mediated by its interaction with the promoter of the alpha-globin gene, some increase in activity is seen when structural elements from the 5' end of the alpha-globin gene are included with the target promoter. In addition, we show that the enhancing activity of the alpha LCR is potentiated by hemin-induction of K562 cells. Whereas phorbol esters that induce megakaryocytic differentiation of K562 cells markedly decrease alpha-globin messenger RNA accumulation, they do not seem to have a negative effect on the activity of the alpha LCR. These studies suggest a role for the alpha LCR in the basal activity of the alpha-globin gene in erythroid cells and in its increased expression seen with erythroid differentiation. The mechanism of negative regulation of alpha-globin gene expression in phorbol-differentiated K562 cells does not appear to be mediated through the action of the alpha LCR.

Base Sequence↗
PubMed1993

Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer.

The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut-NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single-copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

Binding Sites↗
PubMed1996

Erythroid AP-1/NF-E2 elements vary in their response to NF-E2.

AP-1/NF-E2 motifs found in erythroid transcription control elements are associated with powerful transcription activation and thought to be regulated by the erythroid transcription factor NF-E2. We have studied AP-1/NF-E2 motifs from three different erythroid control elements (5'HS2 of the human beta-globin locus control region [LCR], the porphobilinogen deaminase [PBGD] promoter, and the mouse Band 3 promoter). We find that these AP-1/NF-E2 elements differ both in their ability to bind NF-E2 and their activity in transient assays. Each of the elements is bound by AP-1, but only the 5'HS2 and PBGD sites are bound by NF-E2. We examined the activity of these sites in minimal promoter constructs in transient assays. In erythroid cells, activity of duplicated NF-E2 motifs is positively correlated with binding by NF-E2; however, the Band 3 element not bound by NF-E2 is also active in some contexts. In HeLa cells, all sites were active and duplicated sites were most active. In F9 mouse teratocarcinoma cells, which express neither NF-E2 nor AP-1, the elements' activity parallels that in erythroid cells. While these finding are consistent with other evidence that NF-E2 is an important regulator of erythroid transcription, they suggest that some sites that resemble NF-E2 elements are actually regulated by other factors; we speculate that other tissue-specific and/or generally expressed factors may act on these sites.

Animals↗
PubMed1996

Fetal hemoglobin in sickle cell anemia: relation to regulatory sequences cis to the beta-globin gene. Multicenter Study of Hydroxyurea.

Very different fetal hemoglobin levels among adult sickle cell anemia patients suggest genetic modulation of gamma-globin gene expression. In sickle cell anemia, different fetal hemoglobin levels are associated with distinct beta-globin gene haplotypes. Haplotype may be a marker for linked DNA that modulates gamma-globin gene expression. From 295 individuals with sickle cell anemia, we chose for detailed studies 53 patients who had the highest or the lowest fetal hemoglobin levels and 7 patients whose fetal hemoglobin levels were atypical of their haplotype. In these individuals, we examined portions of the beta-globin gene locus control region hypersensitive sites two and three, an (AT)x(T)y repeat 5' to the beta-globin gene, a 4-bp deletion 5' to the A gamma T gene, promoters of both gamma-globin genes, 5' flanking region of the G gamma-globin gene, and A gamma-globin gene IVS-II. Of the regions we studied all polymorphisms were always haplotype-linked and no additional mutations were present. This suggested that variations in these areas are uncommon mechanisms of fetal hemoglobin modulation in sickle cell anemia. Whereas unexamined cis-acting sequences may regulate gamma-globin gene transcription, trans-acting factors may play a more important role.

Anemia, Sickle Cell↗
PubMed1996

Adeno-associated virus 2-mediated transduction and erythroid cell-specific expression of a human beta-globin gene.

Recombinant adeno-associated virus 2 (AAV) virions were constructed that contained the genomic copy of a normal human beta-globin gene marked with a 4-bp Clal linker, and the herpesvirus thymidine kinase (TK) promoter-driven bacterial gene for resistance to neomycin (v beta m-globin), as well as those containing the DNase l-hypersensitive site 2 (HS-2) from the locus control region (LCR) of the human beta-globin gene cluster (vHS2-beta m-globin). These recombinant virions were used to infect a human erythroleukemia cell line which normally does not express the beta-globin gene (K562), or a human nasopharyngeal carcinoma cell line (KB). Cell populations resistant to G418, a neomycin analogue, were obtained following infections with the recombinant virions, indicating high-efficiency transduction of the chimeric gene as well as functional activity of the transduced neo gene in both cell types. Southern blot analysis using a human beta-globin DNA probe substantiated stable integration of the exogenous beta-globin allele in these cells. There was no expression of the transduced beta-globin gene in K562 or KB cells infected with the v beta m-globin virus. High-level expression of the transduced beta-globin gene occurred only in the vHS2-beta m-globin virus-infected K562 cells, but not in KB cells, as determined by Northern blot as well as RNase protection analyses. Expression of the human beta-globin protein could also be detected in approximately 10-20% of the vHS2-beta m-globin virus-infected K562 cells. These studies suggest that the AAV-based vector system may prove useful for high-efficiency globin gene transfer in human hematopoietic cells.

Dependovirus↗
PubMed1996

Developmental regulation of the vertebrate globin multigene family.

"Hemoglobin switching," or the sequential expression of globin genes in erythroid cells during development, has provided an important paradigm for tissue- and stage-specific gene regulation. Over the past decade, regulatory DNA sequences and transcription factors involved in controlling the expression of individual globin genes in erythroid cells have been identified. The picture that has emerged indicates that gene proximal control elements collaborate with a "locus control region" located far upstream - probably via a DNA looping mechanism - to ensure that each gene is turned on only in erythroid cells and at the appropriate time during development. Interactions among the various regulatory sequences are thought to be mediated and stabilized by an array of tissue-specific and ubiquitous proteins. Chromatin structure plays a critical but still poorly understood role in this process.

Chromatin↗
PubMed1997

High-level globin gene expression mediated by a recombinant adeno-associated virus genome that contains the 3' gamma globin gene regulatory element and integrates as tandem copies in erythroid cells.

Recombinant adeno-associated virus (rAAV) vectors are being evaluated for gene therapy applications. Using purified rAAV containing a mutationally marked globin gene (A(gamma)*) and sites 2, 3, and 4 from the locus control region (rHS432A(gamma)*), but lacking a drug-resistance gene, we investigated the relationship between multiplicity of infection (MOI), gene expression, and unselected genome integration in erythroid cells. Most primary erythroid progenitors were transduced as reflected by A(gamma)* mRNA in mature colonies but only at an MOI of greater than 5 x 10(7). Using immortalized erythroleukemia cells as a model, we found that fewer than one half of the colonies that contained the A(gamma)* transcript had an integrated, intact rHS432A(gamma)* genome. rHS432A(gamma)* integrated as a single copy with expression at approximately 50% the level of an endogenous gamma globin gene. A second vector, rHS32A(gamma)*3'RE, containing the regulatory element (RE) from 3' to the chromosomal A(gamma) globin gene, integrated as an intact, tandem head to tail concatamer with a median copy number of 6 with variable expression per copy ranging from approximately onefold to threefold that of an endogenous y globin gene. These results establish that purified rAAV can be used to achieve integration and functional expression of a globin gene in erythroid cells, but only when high MOIs are used.

Dependovirus↗