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PubMed1996

Concerted repression of an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2).

The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus-controlling region, is highly responsive to any of several participating factors.

Animals↗
PubMed1999

Targeting a SWI/SNF-related chromatin remodeling complex to the beta-globin promoter in erythroid cells.

Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human beta-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the beta-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the beta-globin promoter. Because E-RC1 cannot remodel chromatin over the beta-globin promoter without EKLF in vitro, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the ProteIN POsition Identification with Nuclease Tail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the beta-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the beta-globin promoter in mouse erythroleukemia cells. When the beta-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers.

Base Sequence↗
PubMed1999

Mutually exclusive expression of human red and green visual pigment-reporter transgenes occurs at high frequency in murine cone photoreceptors.

This study examines the mechanism of mutually exclusive expression of the human X-linked red and green visual pigment genes in their respective cone photoreceptors by asking whether this expression pattern can be produced in a mammal that normally carries only a single X-linked visual pigment gene. To address this question, we generated transgenic mice that carry a single copy of a minimal human X chromosome visual pigment gene array in which the red and green pigment gene transcription units were replaced, respectively, by alkaline phosphatase and beta-galactosidase reporters. As determined by histochemical staining, the reporters are expressed exclusively in cone photoreceptor cells. In 20 transgenic mice carrying any one of three independent transgene insertion events, an average of 63% of expressing cones have alkaline phosphatase activity, 10% have beta-galactosidase activity, and 27% have activity for both reporters. Thus, mutually exclusive expression of red and green pigment transgenes can be achieved in a large fraction of cones in a dichromat mammal, suggesting a facile evolutionary path for the development of trichromacy after visual pigment gene duplication. These observations are consistent with a model of visual pigment expression in which stochastic pairing occurs between a locus control region and either the red or the green pigment gene promotor.

Animals↗
PubMed1995

cAMP-dependent protein kinase is necessary for increased NF-E2.DNA complex formation during erythroleukemia cell differentiation.

When murine erythroleukemia (MEL) cells are induced to differentiate by hexamethylene bisacetamide (HMBA), erythroid-specific genes are transcriptionally activated; however, transcriptional activation of these genes is severely impaired in cAMP-dependent protein kinase (protein kinase A)-deficient MEL cells. The transcription factor NF-E2, composed of a 45-kDa (p45) and an 18-kDa (p18) subunit, is essential for enhancer activity of the globin locus control regions (LCRs). DNA binding of NF-E2 and alpha-globin LCR enhancer activity was significantly less in HMBA-treated protein kinase A-deficient cells compared to cells containing normal protein kinase A activity; DNA binding of several other transcription factors was the same in both cell types. In parental cells, HMBA treatment and/or prolonged activation of protein kinase A increased the amount of NF-E2.DNA complexes without change in DNA binding affinity; the expression of p45 and p18 was the same under all conditions. p45 and p18 were phosphorylated by protein kinase A in vitro, but the phosphorylation did not affect NF-E2.DNA complexes, suggesting that protein kinase A regulates NF-E2.DNA complex formation indirectly, e.g. by altering expression of a regulatory factor(s). Thus, protein kinase A appears to be necessary for increased NF-E2.DNA complex formation during differentiation of MEL cells and may influence erythroid-specific gene expression through this mechanism.

Base Sequence↗
PubMed1995

Inhibition of an erythroid differentiation switch by the helix-loop-helix protein Id1.

The Id proteins function as negative regulators of basic-helix-loop-helix transcription factors, which play important roles in determination of cell lineage and in tissue-specific differentiation. Down-regulation of Id1 mRNA is associated with dimethyl sulfoxide-induced terminal differentiation of mouse erythroleukemia cells. To examine the significance of Id1 down-regulation in erythroid differentiation, we generated stable mouse erythroleukemia cell lines that constitutively express a "marked" form of the murine Id1 gene. Terminal erythroid differentiation was inhibited in these lines, as indicated by a block in activation of the erythroid-specific genes alpha-globin, beta-globin, and band 3 and continued proliferation in the presence of dimethyl sulfoxide. Interestingly, this block occurred even in the presence of normal levels of the lineage-specific transcription factors GATA-1, NF-E2, and EKLF. Constitutive expression of Id1 did not interfere with DNase I hypersensitivity at site HS2 of the locus control region, expression of the erythropoietin receptor gene, or down-regulation of the endogenous Id1 or c-myc genes. The differentiation block is reversible in these lines and can be rescued by fusion with human erythroleukemia cells. These findings suggest that in vivo, Id1 functions as an antagonist of terminal erythroid differentiation.

Amino Acid Sequence↗
PubMed1996

CAAT/enhancer-binding proteins are involved in beta-globin gene expression and are differentially expressed in murine erythroleukemia and K562 cells.

Acting in cis with the beta-globin locus control region, the CAAT box of the beta-globin gene promoter stimulates transcription 10-fold in murine erythroleukemia (MEL) cells but is without effect in K562 cells. Our previous studies suggested that of four proteins from MEL cells that bind to this CAAT box region (CP1, GATA-1, and two factors that were denoted DSFr and DSF1) DSFr is involved in the up-regulation of transcription. In the present report, the DSFr protein in MEL cells was identified as C/EBPgamma through expression cloning and antibody studies. C/EBPgamma DNA binding activity could not be detected in K562 cells. However, K562 cells, but not MEL cells, were found to express LIP, which is a truncated form of C/EBPbeta and is an inhibitor of transcription. Thus, the differential expression of C/EBP members could account for the ability of the beta-globin CAAT box to stimulate transcription in MEL cells, but not function in K562 cells. Juxtaposing a specific C/EBP binding sequence next to the beta-globin promoter, in constructs in which the CAAT box had been rendered inactive by mutation or deletion, restored full promoter activity in MEL cells only if CP1 still bound to the promoter. In conjunction with previous mutation analyses, these results suggest that C/EBPgamma may collaborate with CP1 to enhance transcription through the beta-globin CAAT box.

Animals↗
PubMed1997

hMAF, a small human transcription factor that heterodimerizes specifically with Nrf1 and Nrf2.

A 1.6-kilobase pair full-length cDNA encoding a transcription factor homologous to the Maf family of proteins was isolated by screening a K562 cDNA library with the NFE2 tandem repeat probe derived from the globin locus control region. The protein, which was designated hMAF, contains a basic DNA binding domain and an extended leucine zipper but lacks any recognizable activation domain. Expressed in vitro, the hMAF protein is able to homodimerize in solution and band-shift the NFE2 tandem repeat probe. In addition to homodimers, hMAF can also form high affinity heterodimers with two members of the NFE2/CNC-bZip family (Nrf1 and Nrf2) but not with a third family member, p45-NFE2. Although hMAF/hMAF homodimers and hMAF/Nrf1 and hMAF/Nrf2 heterodimers bind to the same NFE2 site, they exert functionally opposite effects on the activity of a linked gamma-globin gene. In fact, whereas all hMAF/CNC-bZip heterodimers stimulate the activity of a gamma-promoter reporter construct in K562 cells, the association into homodimers that is induced by overexpressing hMAF inhibits the activity of the same construct. Thus variations in the expression of hMAF may account for the modulation in the activity of the genes that bear NFE2 recognition sites.

Amino Acid Sequence↗
PubMed1998

Maximal activity of an erythroid-specific enhancer requires the presence of specific protein binding sites in linked promoters.

High level expression of many eukaryotic genes is achieved through the action of distal regulatory sequences or enhancers. We have utilized the interaction between the erythroid-specific enhancer in hypersensitivity site 2 (HS2) of the human beta-globin locus control region and the globin gene promoters as a model to elucidate the mechanisms governing promoter/enhancer interactions. HS2 contains a 400-base pair core element consisting of tandem AP1/NF-E2 motifs flanked by binding sites for multiple ubiquitous and erythroid-specific factors. We have compared the enhancer activity of this core element with a synthetic enhancer lacking the factor binding sites flanking the AP1/NF-E2 motif (HS2(M)). In fetal/erythroid K562 cells, enhancement of a linked gamma-promoter was significantly greater with wild-type HS2 than with HS2(M). In contrast, the increase in beta-promoter activity in these cells was equivalent with either enhancer fragment. Truncation of the binding site for the fetal/erythroid-specific stage selector protein in the gamma-promoter abolished the additional enhancer activity of HS2. Similarly, insertion of the stage selector protein site into the beta-promoter boosted enhancer activity observed with HS2 but not HS2(M). In adult erythroid MEL cells, enhancement of a linked beta-promoter was significantly greater with HS2 than with HS2(M). This effect was dependent on the binding of the adult stage-specific factor, erythroid Kruppel-like factor, to the beta-promoter. Taken together, this data suggests that the stage-specific factors binding the proximal globin promoters and the factors flanking the AP1/NF-E2 motif of HS2 act in synergy.

Binding Sites↗
PubMed2000

An Alu element from the K18 gene confers position-independent expression in transgenic mice.

We have identified a 323-base pair fragment of the 5'-flanking sequence of the K18 gene, which confers position-independent and copy number-dependent expression on two heterologous transgenes. This fragment is composed primarily of an Alu repetitive element. Its activity in mice is correlated with its RNA polymerase III promoter activity and its orientation-dependent ability to inhibit potential transcriptional interference in a transfection assay. However, the activity of the Alu element is not correlated with its enhancer blocking activity, a characteristic of insulator elements. In addition, this Alu element did not block the suppressive effect of co-injecting mouse alpha satellite DNA with the transgene. This Alu element is likely responsible for at least part of the protective effects of the sequences flanking the K18. These results suggest that transcriptionally active Alu elements may eliminate transcriptional interference of neighboring genes. This Alu element is one component of the locus control region associated with the K18 gene. Other Alu repetitive elements may also function to define regulatory domains.

Alkaline Phosphatase↗
PubMed2000

Novel transactivation domain in erythroid Kruppel-like factor (EKLF).

Erythroid Kruppel-like Factor (EKLF) is an erythroid-specific transcription factor that plays a critical role in gamma- to beta-globin gene switching during development. To identify essential domains required for EKLF transactivation function, we cotransfected a human erythroleukemia cell line (K562) with a locus control region gamma/Luc-beta/Cat reporter and an EKLF expression vector. In this assay EKLF mediates a 500-fold induction of beta/CAT expression compared with controls. To map essential transactivation domains, progressive NH(2)-terminal and internal deletion mutants of EKLF were constructed. All EKLF mutants were expressed at wild-type levels, localized to the nucleus, and bound DNA. When mutant EKLF proteins were tested for beta/CAT activation, a novel transactivation domain was identified. This novel domain, encompassing amino acids (aa) 140-358, is sufficient for maximal beta/CAT activation. An 85-amino acid subdomain within this region (aa 140-225) is essential for its activity. Interestingly, this central transactivation subdomain is functionally redundant with the amino-terminal domain (aa 1-139). Thus, EKLF possesses at least two potent transactivation domains that appear to function in a redundant manner.

Animals↗
PubMed2003

A WW domain-binding motif within the activation domain of the hematopoietic transcription factor NF-E2 is essential for establishment of a tissue-specific histone modification pattern.

Histone H3 methylated at lysine 4 (H3-meK4) co-localizes with hyperacetylated histones H3 and H4 in transcriptionally active chromatin, but mechanisms that establish H3-meK4 are poorly understood. Previously, we showed that the hematopoietic-specific activator NF-E2, which is required for beta-globin transcription in erythroleukemia cells, induces histone H3 hyperacetylation and H3-meK4 at the adult beta-globin genes (betamajor and betaminor). Chromatin immunoprecipitation analysis indicated that NF-E2 occupies hypersensitive site two (HS2) of the beta-globin locus control region. The mechanism of NF-E2-mediated chromatin modification was investigated by complementation analysis in NF-E2-null CB3 erythroleukemia cells. The activation domain of the hematopoietic-specific subunit of NF-E2 (p45/NF-E2) contains two WW domain-binding motifs (PXY-1 and PXY-2). PXY-1 is required for activation of beta-globin transcription. Here, we determined which step in NF-E2-dependent transactivation is PXY-1-dependent. A p45/NF-E2 mutant lacking 42 amino acids of the activation domain, including both PXY motifs, and a mutant lacking only PXY-1 were impaired in inducing histone H3 hyperacetylation, H3-meK4, and RNA polymerase II recruitment. The PXY motifs were not required for transactivation in the context of a GAL4 DNA-binding domain fusion to p45/NF-E2 in transient transfection assays. As the PXY-1 mutant occupied HS2 normally, the chromatin modification defect occurred post-DNA binding. PXY-1 was not required for recruitment of the histone acetyltransferases cAMP-responsive element-binding protein-binding protein (CBP) and p300 to HS2. These results indicate that PXY-1 confers chromatin-specific transcriptional activation via interaction with a co-regulator distinct from CBP/p300 or by regulating CBP/p300 function.

Acetylation↗
PubMed2005

CHX10 targets a subset of photoreceptor genes.

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.

Animals↗
PubMed2006

Differential binding of erythroid Krupple-like factor to embryonic/fetal globin gene promoters during development.

The competition model for beta-like globin gene switching during development predicts that differential binding of transcription factors to globin gene promoters and/or proximal enhancers regulate the competitive interactions of globin gene family members with the powerful locus control region (LCR). Direct interactions of individual genes with the LCR are essential for high level expression in erythroid cells. In this paper, we have demonstrated, by chromatin immunoprecipitation, 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 3-fold higher than the level in primitive cells. These results suggest that temporal-specific 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.

Animals↗
PubMed1994

Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood.

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.

Antigens, CD↗
PubMed1998

Interleukin (IL)-4-independent induction of immunoglobulin (Ig)E, and perturbation of T cell development in transgenic mice expressing IL-13.

Recent studies using interleukin (IL)-4-deficient animals have highlighted the existence of IL-4-independent immunoglobulin (Ig)E induction. We have established transgenic mice expressing IL-13 from a transgene comprising a genomic fragment containing the IL-13 gene and the human CD2 locus control region. The transgenes were expressed in lymphoid tissues and induced by T cell activators, suggesting regulation by elements of the IL-13 promoter. IL-13 transgenic lines expressed 10-100-fold higher levels of serum IgE than their littermate controls, but had normal levels of other serum Ig isotypes. Elevated IgE levels were also detected in sera from IL-4-deficient mice carrying IL-13 transgenes, indicating that IL-4 is not required for IL-13-induced IgE expression in the mouse. Expression of IL-13 also perturbed the development of thymocytes. Although thymocyte development was normal up to 4 wk of age, thymocyte number decreased dramatically thereafter, reaching 10% of normal by 10 wk, and despite normal size and appearance, histological examination demonstrated that transgenic thymi contained only small foci of thymocytes. The reduction in thymocyte number was due mainly to a depletion of CD4(+)CD8(+) thymocytes, and did not affect significantly the composition of peripheral T cell populations. These data indicate that expression of IL-13 transgenes in vivo can regulate IgE production in the mouse, and that IL-13 may also influence thymocyte development.

Animals↗
PubMed2003

High-level factor VIII gene expression in vivo achieved by nonviral liver-specific gene therapy vectors.

Two liver-specific nonviral gene transfer vectors have been developed to accommodate heterologous genes. The expression cassettes contain (1) a hepatic locus control region from the apolipoprotein E (ApoE) gene (HCR), (2) a liver-specific alpha(1)-antitrypsin promoter (HP), (3) a 1.4-kb truncated factor IX first intron (I) or a synthetic minx intron (mI), (4) a multiple cloning site (MCS) for inserting cDNA sequences, and (5) a bovine growth hormone polyadenylation signal (bpA) to make pBS-HCRHPI-A or pBS-HCRHPmI-A. These vectors were first evaluated with reporter genes encoding human factor IX (hFIX) and green fluorescent protein (GFP). hFIX constructs, pBS-HCRHPI-FIXA and control pBS-HCRHP-FIXIA with the hFIX intron in its native position, produced comparable hFIX gene expression levels (0.5-5 microg/ml) 6 months after naked DNA transfer to mice, whereas the factor IX level from pBS-HCRHPmI-FIXA averaged about 50% lower. RT-PCR analysis of the mRNA indicated that introns inserted upstream from the cDNA were correctly processed and spliced. GFP expression was detected in 15-30% of the hepatocytes in pBS-HCRHPI-GFPA-treated mice. Next, a B domain-deleted human factor VIII (hFVIII) cDNA was inserted into the modified vectors. High-level hFVIII expression (up to 750 ng/ml) was achieved initially in both C57BL/6 mice and Rag2 mice. Moreover, therapeutic levels of hFVIII (20-310 ng/ml) circulated in Rag2 mice 6 months after treatment. These liver-specific gene expression cassettes can deliver a large, heterologous gene such as hFVIII cDNA to achieve high-level, persistent transgene expression after in vivo hepatic gene therapy.

Animals↗
PubMed2003

Evidence that the mouse 3' kappa light chain enhancer confers position-independent transgene expression in T- and B-lineage cells.

One of the major obstacles for successful application of murine leukemia virus (MLV) vectors to genetic therapy of lymphocyte disorders is low levels of transgene expression or the eventual loss of expression. To overcome this problem, an improved retroviral vector was constructed utilizing the myeloproliferative sarcoma virus (MPSV) long terminal repeat (LTR), which provided a significantly higher level of transgene expression in human lymphoid cells than did MLV vectors. Nevertheless, transgene expression remained low in a large percentage of transduced cells. To address whether lymphocyte enhancer elements might improve transgene expression mediated by retroviral vectors in lymphocytes, we cloned the mouse immunoglobulin 3' kappa light chain enhancer gene (mE3') into the MPSV vector. We found that the mE3' conferred a higher, more uniform and sustained level of expression in transduced T- and B-cell lines, and in primary T cells, than did the control vector lacking this element. Integration sites were diverse and a single copy of the proviral genome was present in all examined transduced cells. The mE3' failed to enhance transgene expression in most nonlymphoid cells, indicating it is relatively lineage-specific. Taken together, these results provide strong evidence that the mE3' functions as a locus control region (LCR) in conferring enhanced integration-site-independent expression of a retroviral transgene.

Animals↗
PubMed1999

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted expression from parvovirus B19p6 promoter in primary human hematopoietic progenitor cells.

Human parvovirus B19 gene expression from the viral p6 promoter (B19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. We have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (Ponnazhagan et al. J Virol 69:8096-8101, 1995). However, abundant expression from this promoter can be readily detected in primary human bone marrow cells (Wang et al. Proc Natl Acad Sci USA 92:12416-12420, 1995; Ponnazhagan et al. J Gen Virol 77:1111-1122, 1996). In the present studies, we investigated the pattern of expression from the B19p6 promoter in primary human bone marrow-derived CD34+ HPC undergoing differentiation into myeloid and erythroid lineages. CD34+ cells were transduced with recombinant adeno-associated virus 2 (AAV) vectors containing the beta-galactosidase (lacZ) gene under the control of the following promoters/enhancers: the cytomegalovirus promoter (vCMVp-lacZ), B19p6 promoter (vB19p6-lacZ), B19p6 promoter with an upstream erythroid cell-specific enhancer element (HS-2) from the locus control region (LCR) from the human beta-globin gene cluster (vHS2-B19p6-lacZ), and the human beta-globin gene promoter with the HS-2 enhancer (vHS2-beta p-lacZ). Transgene expression was evaluated either 48 h after infection or following erythroid differentiation in vitro for 3 weeks. Whereas high-level expression from the CMV promoter 48 h after infection diminished with time, low-level expression from the B19p6 and the beta-globin promoters increased significantly following erythroid differentiation. Furthermore, in HPC assays, there was no significant difference in the level of expression from the CMV promoter in myeloid or erythroid cell-derived colonies. Expression from the B19p6 and the beta-globin promoters, on the other hand, was restricted to erythroid cell colonies. These data further corroborate that the B19p6 promoter is erythroid cell-specific and suggest that the recombinant AAV-B19 hybrid vectors may prove useful in gene therapy of human hemoglobinopathies in general and sickle cell anemia and beta-thalassemia in particular.

Anemia, Sickle Cell↗