Spatial organization of the chicken beta-globin gene domain in erythroid cells of embryonic and adult lineages

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

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A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽

10.1182/blood.v81.5.1384.bloodjournal8151384 ◽

1993 ◽

Vol 81 (5) ◽

pp. 1384-1392

Author(s):

I Plavec ◽

T Papayannopoulou ◽

C Maury ◽

F Meyer

Keyword(s):

Bone Marrow Cells ◽

Globin Gene ◽

Beta Globin ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Beta Globin Gene ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia ◽

In Erythroid Cells

Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

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5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4690 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4690-4697 ◽

Cited By ~ 74

Author(s):

J G Glauber ◽

N J Wandersee ◽

J A Little ◽

G D Ginder

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Globin Gene ◽

Beta Globin ◽

Erythroid Cells ◽

Adult Chicken ◽

Beta Globin Gene ◽

Flanking Sequences ◽

Murine Erythroleukemia ◽

Globin Gene Expression

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

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The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4958-4965.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4958-4965

Author(s):

V Dhar ◽

D Mager ◽

A Iqbal ◽

C L Schildkraut

Keyword(s):

Cell Lines ◽

Dna Sequences ◽

Globin Gene ◽

K562 Cells ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Hypersensitive Sites ◽

Flanking Sequences ◽

Globin Gene Domain

The temporal order of replication of DNA sequences in the chromosomal domain containing the human beta-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the beta-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The beta-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the beta-globin domain.

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Erythroleukemia (K562) cells contain a functional beta-globin gene

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2553-2555.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2553-2555

Author(s):

M Donovan-Peluso ◽

K Young ◽

C Dobkin ◽

A Bank

Keyword(s):

Hela Cells ◽

Globin Gene ◽

K562 Cells ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene ◽

Rna Transcripts

K562 cells are human erythroid cells that synthesize embryonic and fetal globins but not adult beta-globin. A cloned beta-globin gene was isolated from K562 cells and transfected into HeLa cells. The RNA transcripts produced were comparable in both amount and size to those obtained with a normal beta-globin gene.

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Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.7.11.4024-4029.1987 ◽

1987 ◽

Vol 7 (11) ◽

pp. 4024-4029

Author(s):

M Trudel ◽

J Magram ◽

L Bruckner ◽

F Costantini

Keyword(s):

Transgenic Mice ◽

Fusion Gene ◽

Globin Gene ◽

Regulatory Elements ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Base Pairs ◽

Beta Globin Gene ◽

Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

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Human Beta-Globin Expression during Erythroid Cells Development in Transgenic Mice Bearing Human Beta-Globin Gene with Mutated BP1 Binding Site.

Blood ◽

10.1182/blood.v106.11.3651.3651 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3651-3651

Author(s):

Olga P. Zoueva ◽

David Bodine ◽

Griffin P. Rodgers

Keyword(s):

Transgenic Mice ◽

Binding Site ◽

Fetal Liver ◽

Globin Gene ◽

Fold Increase ◽

Mouse Embryos ◽

Mrna Levels ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene

Abstract Binding of beta protein 1 (BP1) to its site on the promoter of adult beta-globin gene has silencing effect on beta-globin transcription in vitro. To better understand the mechanism of the negative regulation of beta-globin expression by BP1 we have developed transgenic mice. Specifically, we introduced a mutated BP1 binding site into the promoter of beta-globin gene sequence of 35 kb cosmid construct. This construct containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into the single cell mouse embryos. To detect the differences in developmental regulation of the human beta-globin gene expression in the transgenic mice, we studied the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5. In addition, we analyzed adult erythroid cells. To minimize experimental error, samples from individual animals of three transgenic lines were analyzed independently using real-time PCR assays. Levels of expression of murine alpha-globin mRNA were used as internal controls. The BP1 gene and its mouse analog Dlx4 belongs to the Distal-less family of homeobox genes, which are expressed during early development. We found that the mRNA levels of human beta-globin in transgenic mice containing mutated BP1 binding site were higher at all stages of erythroid cells development as compared with control transgenic mice bearing cosmid construct with wild type sequence of BP1 site. Particularly, we detected up to 20-fold increase in human beta-globin expression in embryonic blood at E10.5, 3-fold increase in fetal livers of transgenic mice at E13.5, and up to 1.4-fold increase in adult reticulocytes. We also found that increase in human beta-globin expression was correlated with expression pattern of murine Dlx4 which mRNA was predominantly expressed in embryonic blood at E10.5. Thus, our data indicate that transgenic mice bearing human beta-globin gene with mutated BP1 site have significantly higher human beta-globin transcripts levels in blood cells from primitive erythropoesis than control mice. These results may help develop the novel clinic approaches for the inhibition of the expression of abnormal beta-globin genes, such as sickle (hbs) and hbc.

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Erythroleukemia (K562) cells contain a functional beta-globin gene.

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2553 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2553-2555 ◽

Cited By ~ 10

Author(s):

M Donovan-Peluso ◽

K Young ◽

C Dobkin ◽

A Bank

Keyword(s):

Hela Cells ◽

Globin Gene ◽

K562 Cells ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene ◽

Rna Transcripts

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Altered Regulation of β like Globin Genes by a Redesigned Erythroid Transcription Factor.

Blood ◽

10.1182/blood.v104.11.1212.1212 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1212-1212

Author(s):

Deepa Manwani ◽

Mariann Galdass ◽

James J. Bieker

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Zinc Finger ◽

Embryoid Body ◽

Globin Gene ◽

Beta Globin ◽

Erythroid Cells ◽

Beta Globin Gene ◽

Body Development ◽

Globin Promoter

Abstract The well characterized switch during ontogeny of globin gene expression from embryonic/ fetal to adult type is a result of a complex interplay between cis and trans acting regulatory elements at the beta globin locus. Trans acting elements include tissue specific transcription factors that bind specific motifs within the beta globin gene cluster with high specificity. Erythroid Kruppel like factor (EKLF) is one such erythroid specific, zinc finger transcription factor that is critical for the activation of the beta globin promoter and for consolidating the switch from gamma to beta globin during development. The ability to willfully regulate the expression of endogenous genes using redesigned zinc finger transcription factors is an emerging field. There is tremendous appeal in utilizing the understanding of transcriptional control pathways to design tools that will elucidate molecular mechanisms and provide potential therapeutic tools. To this end we redesigned Erythroid Kruppel Like Factor (EKLF) as a transcriptional repressor. The zinc finger DNA binding domain was linked to the repressor domain from the Drosophila Engrailed protein with the prediction that this construct (ENG/ZNF) would bind the beta globin promoter and repress it. It was hypothesized that embryonic/fetal globin activation would result by a competitive mechanism. When introduced transiently into cells these transcription factors are effective in repressing the adult beta globin promoter CACCC element, the natural target for EKLF. In stable MEL clones, repression of the adult beta globin gene is accompanied by a reactivation of the endogenous embryonic globin gene. In order to study this effect in the context of a whole animal we generated transgenic mice expressing ENG/ZNF. A 271 bp region 5′of the ANK-1 gene was chosen to drive expression in transgenic mice as it provides erythroid specific expression with copy number dependence and minimal position dependence. D13.5 fetal livers were subject to RT-PCR analysis in the linear range to quantitate the ratios of BH1 to alpha globin transcripts. The 9 ENG/ZNF transgenic embryos express BH1 mRNa in a range of values that is statistically higher than in 9 control littermates (Mann Whitney U test, p value 0.02) and beta major globin mRNA at lower levels. We further studied ENG/ZNF in the developmentally plastic environment of differentiating murine embryonic stem cells. The construct was stably integrated into a targeting site upstream of the HPRT locus under the control of a tetracycline inducible promoter. The Doxycycline induction of ENG/ZNF transgene expression results in a 4 fold activation of embryonic globin at day 6 of embryoid body development; however there is no evidence of beta globin repression. Since at this stage of embryoid body development, primitive erythroid cells are 100–500 fold more abundant than definitive erythroid cells, this may reflect a differential effect of EKLF in primitive erythroid cells. To evaluate this further, we are currently performing analyses in primitive versus definitive erythroid colonies. In conclusion, our studies support the competitive model of globin switching and may contribute to the delineation of a stage specific role of EKLF. In addition, transcriptional reagents that augment gamma globin expression hold promise as novel therapeutic agents for sickle cell disease and other hemoglobinopathies.

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Signals in chicken beta-globin DNA influence chromatin assembly in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7596 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7596-7603 ◽

Cited By ~ 7

Author(s):

K Liu ◽

J D Lauderdale ◽

A Stein

Keyword(s):

Intergenic Region ◽

Globin Gene ◽

Linker Histone ◽

Chromatin Assembly ◽

Beta Globin ◽

Erythroid Cells ◽

Core Histones ◽

Histone H5 ◽

In Erythroid Cells

We have confirmed the result that chicken beta-globin gene chromatin, which possesses the characteristics of active chromatin in erythroid cells, has shortened internucleosome spacings compared with bulk chromatin or that of the ovalbumin gene, which is inactive. To understand how the short (approximately 180-bp) nucleosome repeat arises specifically on beta-globin DNA, we have studied chromatin assembly of cloned chicken beta-globin DNA in a defined in vitro system. With chicken erythrocyte core histones and linker histone H5 as the only cellular components, a cloned 6.2-kb chicken beta-globin DNA fragment assembled into chromatin possessing a regular 180 +/- 5-bp repeat, very similar to what is observed in erythroid cells. A 2-kb DNA subfragment containing the beta A gene and promoter region, but lacking the downstream intergenic region between the beta A and epsilon genes, failed to generate a regular nucleosome array in vitro, suggesting that the intergenic region facilitates linker histone-induced nucleosome alignment. When the beta A gene was placed on a plasmid that contained a known chromatin-organizing signal, nucleosome alignment with a 180-bp periodicity was restored, whereas nucleosomes on flanking plasmid sequences possessed a 210-bp spacing periodicity. Our results suggest that the shortened 180-bp nucleosome spacing periodicity observed in erythroid cells is encoded in the beta-globin DNA sequence and that nucleosome alignment by linker histones is facilitated by sequences in the beta A-epsilon intergenic region.

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