Regulated expression of amplified human beta globin genes

Gene therapy for the beta thalassemias and sickle cell anemia will require high levels of expression of human beta globin genes. One method to achieve this goal is amplification of globin genes transferred into the stem cells in the bone marrow of these patients. If the amplified genes remain normally regulated, they will then further increase their expression on being induced to differentiate along an erythroid pathway. To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human beta globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells. All the G418-resistant clones analyzed acquired and expressed the human beta globin gene. By serial passage of the cells in increasing concentrations of methotrexate, the exogenous human beta globin genes were stably amplified in all lines, and all increased their globin mRNA expression roughly proportional to their augmented copy number. Most of the clones further increased their beta globin expression on addition of an erythroid stimulus (dimethylsulfoxide). These results indicate that globin gene amplification may be useful in increasing globin mRNA expression in further experiments whose goal is gene therapy.

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Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1725-1735.1988 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1725-1735

Author(s):

M A Bender ◽

A D Miller ◽

R E Gelinas

Keyword(s):

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Globin Mrna ◽

Beta Globin Gene ◽

Erythroleukemia Cells ◽

New Gene ◽

Normal Human ◽

Murine Erythroleukemia ◽

Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

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Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1725 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1725-1735 ◽

Cited By ~ 38

Author(s):

M A Bender ◽

A D Miller ◽

R E Gelinas

Keyword(s):

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Globin Mrna ◽

Beta Globin Gene ◽

Erythroleukemia Cells ◽

New Gene ◽

Normal Human ◽

Murine Erythroleukemia ◽

Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

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Fetal hemoglobin silencing in humans

Blood ◽

10.1182/blood-2006-04-015859 ◽

2006 ◽

Vol 108 (6) ◽

pp. 2081-2086 ◽

Cited By ~ 23

Author(s):

Patricia A. Oneal ◽

Nicole M. Gantt ◽

Joseph D. Schwartz ◽

Natarajan V. Bhanu ◽

Y. Terry Lee ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Postnatal Life ◽

Globin Genes ◽

Beta Globin ◽

Globin Mrna ◽

Beta Globin Gene ◽

Gamma Globin ◽

Age Related

Abstract Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

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Differential expression of alpha- and beta-globin genes in erythroleukemic cell lines

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3591-3595.1990 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3591-3595

Author(s):

N Beru ◽

P B Maples ◽

O Hermine ◽

E Goldwasser

Keyword(s):

Cell Lines ◽

Globin Gene ◽

Restriction Enzymes ◽

Erythroid Differentiation ◽

Globin Genes ◽

Beta Globin ◽

Globin Mrna ◽

Beta Globin Gene ◽

Alpha Globin ◽

Erythroleukemic Cell

The IW32, NN10, and IW201 cell lines are erythroleukemic cell lines isolated from the spleens of mice infected with the Friend virus. IW32 and NN10 cells can be induced toward erythroid differentiation and hemoglobin synthesis by hemin or butyrate. Both cell lines contain some mature alpha- and beta-globin mRNA before induction, and addition of the inducers greatly increases the amount of globin message. Unlike IW32 and NN10 cells, IW201 cells are only partially inducible. Uninduced 201 cells contain a small amount of alpha-globin mRNA but no detectable beta-globin message. After induction, the cells contain markedly increased amounts of alpha-globin mRNA but still do not express the beta-globin gene. Southern blot analysis with 10 restriction enzymes shows that the restriction map of the beta-globin gene in IW201 cells is indistinguishable from that in IW32 and NN10 cells.

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Differential expression of alpha- and beta-globin genes in erythroleukemic cell lines.

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3591 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3591-3595 ◽

Cited By ~ 11

Author(s):

N Beru ◽

P B Maples ◽

O Hermine ◽

E Goldwasser

Keyword(s):

Cell Lines ◽

Globin Gene ◽

Restriction Enzymes ◽

Erythroid Differentiation ◽

Globin Genes ◽

Beta Globin ◽

Globin Mrna ◽

Beta Globin Gene ◽

Alpha Globin ◽

Erythroleukemic Cell

The IW32, NN10, and IW201 cell lines are erythroleukemic cell lines isolated from the spleens of mice infected with the Friend virus. IW32 and NN10 cells can be induced toward erythroid differentiation and hemoglobin synthesis by hemin or butyrate. Both cell lines contain some mature alpha- and beta-globin mRNA before induction, and addition of the inducers greatly increases the amount of globin message. Unlike IW32 and NN10 cells, IW201 cells are only partially inducible. Uninduced 201 cells contain a small amount of alpha-globin mRNA but no detectable beta-globin message. After induction, the cells contain markedly increased amounts of alpha-globin mRNA but still do not express the beta-globin gene. Southern blot analysis with 10 restriction enzymes shows that the restriction map of the beta-globin gene in IW201 cells is indistinguishable from that in IW32 and NN10 cells.

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Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3524-3532.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3524-3532

Author(s):

V Dhar ◽

A I Skoultchi ◽

C L Schildkraut

Keyword(s):

Cell Line ◽

Hybrid Cell ◽

Globin Gene ◽

Fibroblast Cell ◽

Globin Genes ◽

Fibroblast Cell Line ◽

Beta Globin ◽

Hybrid Cells ◽

Beta Globin Gene ◽

Cell Hybrids

To investigate whether a switch in the transcriptional activity of a gene is associated with a change in the timing of replication during the S phase, we examined the replication timing of the beta-globin genes in two different types of somatic cell hybrids. In mouse hepatoma (Hepa 1a) x mouse erythroleukemia (MEL) hybrid cells, the beta-globin gene from the MEL parent is transcriptionally inactivated and is later replicating than in the parental MEL cell line. In human fibroblast (GM3552) x MEL hybrid cells, the human beta-globin gene is transcriptionally activated, and all of the sequences within the human beta-globin domain (200 kilobases) we have examined appear to be earlier replicating than those in the parental fibroblast cell line. The chromatin configuration of the activated human beta-globin domain in the hybrids is relatively more sensitive to nucleases than that in the fibroblasts. Furthermore, major nuclease-hypersensitive sites that were absent in the chromatin flanking the distal 5' region of the human beta-globin gene cluster in the parental fibroblast cell line are present in the transcriptionally activated domain in the hybrid cell line. These results suggest that timing of replication of globin genes has been altered in these hybrid cells and thus is not fixed during the process of differentiation.

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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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Molecular analysis of Japanese delta beta-thalassemia

Blood ◽

10.1182/blood.v72.5.1771.bloodjournal7251771 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1771-1776

Author(s):

S Shiokawa ◽

H Yamada ◽

Y Takihara ◽

E Matsunaga ◽

Y Ohba ◽

...

Keyword(s):

Mammalian Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

Large Deletions ◽

Deletion Junction

A DNA fragment containing the deletion junction region from a Japanese individual with homozygous delta beta-thalassemia has been cloned. A clone containing the normal DNA surrounding the 3′ breakpoint of this deletion and a clone carrying the G gamma- and A gamma-globin genes of this patient were also isolated. Sequences of the deletion junction and both gamma-globin genes were determined. A comparison of these sequences with previously determined sequences of the normal counterparts revealed that the 5′ breakpoint is located between 2,134 and 2,137 base pairs (bp) 3′ to the polyA site of the A gamma-globin gene, the 5′ breakpoint is located just downstream of the 3′ border of the fetal gamma-globin duplication unit, and no molecular defects are evident within the gamma-globin gene region. A comparison between the sequences of the normal DNA surrounding the 3′ breakpoint and the normal DNA surrounding the 5′ breakpoint shows that deletion is the result of a nonhomologous recombination event. There are A+T-rich stretches near the 5′ and 3′ breakpoints in the normal DNA, and a portion of an Aly repeat is located in the region 3′ to the 3′ breakpoint. Southern blot analysis using probes 3′ to the beta-globin gene showed that the deletion extends in the 3′ direction further than any other deletions associated with delta beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) heretofore reported. These results are discussed in terms of the mechanism generating large deletions in mammalian cells and three models for the regulation of gamma-globin and beta-globin gene expression in humans.

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Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain

Blood ◽

10.1182/blood.v81.10.2781.bloodjournal81102781 ◽

1993 ◽

Vol 81 (10) ◽

pp. 2781-2790

Author(s):

DE Fleenor ◽

RE Kaufman

Keyword(s):

Topoisomerase Ii ◽

Globin Gene ◽

Dnase I ◽

Globin Genes ◽

Beta Globin ◽

Hypersensitive Site ◽

Mobility Shift ◽

Enhancer Activity ◽

Beta Globin Gene

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.

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