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

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.

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

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.

scholarly journals Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

1994 ◽  
Vol 14 (10) ◽  
pp. 6936-6943 ◽  
Author(s):  
P J Detloff ◽  
J Lewis ◽  
S W John ◽  
W R Shehee ◽  
R Langenbach ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Second Step ◽  
Globin Genes ◽  
Beta Globin ◽  
Es Cell ◽  
Beta Globin Gene ◽  
Targeting Strategy

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

scholarly journals Erythroid-specific nuclease-hypersensitive sites flanking the human beta-globin domain.

1990 ◽  
Vol 10 (8) ◽  
pp. 4324-4333 ◽  
Author(s):  
V Dhar ◽  
A Nandi ◽  
C L Schildkraut ◽  
A I Skoultchi
Keyword(s):  
Dna Sequences ◽  
Chromatin Structure ◽  
Globin Gene ◽  
Embryonal Carcinoma Cell ◽  
Globin Genes ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Specific Expression ◽  
Structure Changes ◽  
Hypersensitive Sites

Recent evidence suggests that DNA sequences from the region lying 5' of the human epsilon-globin gene are important for erythroid-specific expression of human beta-like globin genes. This region, as well as a region 20 kilobases (kb) downstream from the beta-globin gene, contains a set of developmentally stable, DNase I-superhypersensitive sites that are thought to reflect a chromatin structure supporting active globin gene expression. We have analyzed the chromatin structure in these two regions in a wide variety of nonerythroid and erythroid cells. The study included analysis of chromatin structure changes occurring during globin gene activation in mouse erythroleukemia-human nonerythroid cell hybrids. The results identified a hypersensitive site (III) 14.8 kb upstream of the epsilon-globin gene that was strictly correlated with active globin gene transcription. Interestingly, a multipotent human embryonal carcinoma cell line exhibited a hypersensitive site (IV) 18.4 kb upstream of epsilon-globin that was absent in all other nonerythroid cells examined, suggesting that chromatin structure changes at specific hypersensitive sites during embryonic development may also be important in globin gene repression.

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

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.

Erythroid-specific nuclease-hypersensitive sites flanking the human beta-globin domain

1990 ◽  
Vol 10 (8) ◽  
pp. 4324-4333
Author(s):  
V Dhar ◽  
A Nandi ◽  
C L Schildkraut ◽  
A I Skoultchi
Keyword(s):  
Dna Sequences ◽  
Chromatin Structure ◽  
Globin Gene ◽  
Embryonal Carcinoma Cell ◽  
Globin Genes ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Specific Expression ◽  
Structure Changes ◽  
Hypersensitive Sites

Recent evidence suggests that DNA sequences from the region lying 5' of the human epsilon-globin gene are important for erythroid-specific expression of human beta-like globin genes. This region, as well as a region 20 kilobases (kb) downstream from the beta-globin gene, contains a set of developmentally stable, DNase I-superhypersensitive sites that are thought to reflect a chromatin structure supporting active globin gene expression. We have analyzed the chromatin structure in these two regions in a wide variety of nonerythroid and erythroid cells. The study included analysis of chromatin structure changes occurring during globin gene activation in mouse erythroleukemia-human nonerythroid cell hybrids. The results identified a hypersensitive site (III) 14.8 kb upstream of the epsilon-globin gene that was strictly correlated with active globin gene transcription. Interestingly, a multipotent human embryonal carcinoma cell line exhibited a hypersensitive site (IV) 18.4 kb upstream of epsilon-globin that was absent in all other nonerythroid cells examined, suggesting that chromatin structure changes at specific hypersensitive sites during embryonic development may also be important in globin gene repression.

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

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.

scholarly journals Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy

1994 ◽  
Vol 14 (10) ◽  
pp. 6936-6943
Author(s):  
P J Detloff ◽  
J Lewis ◽  
S W John ◽  
W R Shehee ◽  
R Langenbach ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Second Step ◽  
Globin Genes ◽  
Beta Globin ◽  
Es Cell ◽  
Beta Globin Gene ◽  
Targeting Strategy

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

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

1991 ◽  
Vol 11 (9) ◽  
pp. 4690-4697 ◽  
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.

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

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.

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

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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