scholarly journals Active beta-globin gene transcription occurs in methylated, DNase I-resistant chromatin of nonerythroid chicken cells.

1990 ◽  
Vol 10 (1) ◽  
pp. 16-27 ◽  
Author(s):  
R Lois ◽  
L Freeman ◽  
B Villeponteau ◽  
H G Martinson
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Housekeeping Gene ◽  
Dnase I ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Dnase I Sensitivity ◽  
Gene Transcripts ◽  
Per Se ◽  
Termination Process

We report active, inappropriate transcription of the chicken beta A-globin gene in normal fibroblasts, cultured MSB cells, and brain. We were unable to detect ovalbumin gene transcription in these same tissues. Most of the globin gene transcripts were found to be truncated near the beginning of the gene, suggesting the existence of a premature termination process that is preferentially active under conditions of inappropriate transcription. The inappropriately transcribed beta A-globin gene chromatin remained DNase I resistant and highly methylated. Thus, the DNase I-sensitive conformation of erythrocyte beta A chromatin was correlated not with beta A transcription per se but with beta A expression. Although both transcribed and nontranscribed genes within the globin domain exhibited the same DNase I sensitivity in erythrocyte nuclei, a housekeeping gene active in erythrocytes exhibited a different level of DNase I sensitivity. However, this gene exhibited the same level of DNase I sensitivity in both erythrocytes and a cultured cell line. These observations are consistent with the proposal (G. Blobel, Proc. Natl. Acad. Sci. USA 82:8527-8529, 1985) that the DNase I sensitivity of a gene may reflect properties of chromatin related to cotranscriptional and posttranscriptional aspects of mRNA production rather than to transcription per se.

Active beta-globin gene transcription occurs in methylated, DNase I-resistant chromatin of nonerythroid chicken cells

1990 ◽  
Vol 10 (1) ◽  
pp. 16-27
Author(s):  
R Lois ◽  
L Freeman ◽  
B Villeponteau ◽  
H G Martinson
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Housekeeping Gene ◽  
Dnase I ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Dnase I Sensitivity ◽  
Gene Transcripts ◽  
Per Se ◽  
Termination Process

We report active, inappropriate transcription of the chicken beta A-globin gene in normal fibroblasts, cultured MSB cells, and brain. We were unable to detect ovalbumin gene transcription in these same tissues. Most of the globin gene transcripts were found to be truncated near the beginning of the gene, suggesting the existence of a premature termination process that is preferentially active under conditions of inappropriate transcription. The inappropriately transcribed beta A-globin gene chromatin remained DNase I resistant and highly methylated. Thus, the DNase I-sensitive conformation of erythrocyte beta A chromatin was correlated not with beta A transcription per se but with beta A expression. Although both transcribed and nontranscribed genes within the globin domain exhibited the same DNase I sensitivity in erythrocyte nuclei, a housekeeping gene active in erythrocytes exhibited a different level of DNase I sensitivity. However, this gene exhibited the same level of DNase I sensitivity in both erythrocytes and a cultured cell line. These observations are consistent with the proposal (G. Blobel, Proc. Natl. Acad. Sci. USA 82:8527-8529, 1985) that the DNase I sensitivity of a gene may reflect properties of chromatin related to cotranscriptional and posttranscriptional aspects of mRNA production rather than to transcription per se.

Differences in DNase I sensitivity and methylation within the human beta-globin gene domain and correlation with expression

1986 ◽  
Vol 156 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Calliope ARAPINIS ◽  
Jacques ELION ◽  
Dominique LABIE ◽  
Rajagopal KRISHNAMOORTHY
Keyword(s):  
Globin Gene ◽  
Dnase I ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Dnase I Sensitivity ◽  
Globin Gene Domain

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

Blood ◽  
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.

scholarly journals Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo.

1987 ◽  
Vol 7 (5) ◽  
pp. 1917-1924 ◽  
Author(s):  
B Villeponteau ◽  
H G Martinson
Keyword(s):  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Torsional Stress ◽  
Beta Globin ◽  
Cell Penetration ◽  
Dnase I Sensitivity ◽  
Chicken Erythrocytes ◽  
Active Genes

The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA (Villeponteau et al., Cell 39:469-478, 1984). This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

scholarly journals Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

1985 ◽  
Vol 5 (6) ◽  
pp. 1498-1511 ◽  
Author(s):  
P Charnay ◽  
P Mellon ◽  
T Maniatis
Keyword(s):  
Hela Cell ◽  
Gene Transcription ◽  
Hela Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Sequence Element ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Flanking Region ◽  
Mouse Erythroleukemia

We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

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

Blood ◽  
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

Abstract 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.

Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells

1985 ◽  
Vol 5 (6) ◽  
pp. 1498-1511
Author(s):  
P Charnay ◽  
P Mellon ◽  
T Maniatis
Keyword(s):  
Hela Cell ◽  
Gene Transcription ◽  
Hela Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Sequence Element ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Flanking Region ◽  
Mouse Erythroleukemia

We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

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