scholarly journals A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene.

1990 ◽  
Vol 10 (2) ◽  
pp. 528-538 ◽  
Author(s):  
K L Chow ◽  
R J Schwartz
Keyword(s):  
Transcriptional Activity ◽  
Primary Cultures ◽  
Gene Promoter ◽  
Regulatory Sequence ◽  
Actin Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Cell Type Specific Expression ◽  
Alpha Actin

The chicken skeletal alpha-actin gene promoter region provides at least a 75-fold-greater transcriptional activity in muscle cells than in fibroblasts. The cis-acting sequences required for cell type-restricted expression within this 200-base-pair (bp) region were elucidated by chloramphenicol acetyltransferase assays of site-directed Bg/II linker-scanning mutations transiently transfected into primary cultures. Four positive cis-acting elements were identified and are required for efficient transcriptional activity in myogenic cells. These elements, conserved across vertebrate evolution, include the ATAAAA box (-24 bp), paired CCAAT-box-associated repeats (CBARs; at -83 bp and -127 bp), and the upstream T+A-rich regulatory sequence (at -176 bp). Basal transcriptional activity in fibroblasts was not as dependent on the upstream CBAR or regions of the upstream T+A-rich regulatory sequence. Transfection experiments provided evidence that positive regulatory factors required for alpha-actin expression in fibroblasts are limiting. In addition, negative cis-acting elements were detected and found closely associated with the G+C-rich sequences that surround the paired CBARs. Negative elements may have a role in restricting developmentally timed expression in myoblasts and appear to inhibit promoter activity in nonmyogenic cells. Cell type-specific expression of the skeletal alpha-actin gene promoter is regulated by combinatorial and possibly competitive interactions between multiple positive and negative cis-acting elements.

A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene

1990 ◽  
Vol 10 (2) ◽  
pp. 528-538
Author(s):  
K L Chow ◽  
R J Schwartz
Keyword(s):  
Transcriptional Activity ◽  
Primary Cultures ◽  
Gene Promoter ◽  
Regulatory Sequence ◽  
Actin Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Cell Type Specific Expression ◽  
Alpha Actin

The chicken skeletal alpha-actin gene promoter region provides at least a 75-fold-greater transcriptional activity in muscle cells than in fibroblasts. The cis-acting sequences required for cell type-restricted expression within this 200-base-pair (bp) region were elucidated by chloramphenicol acetyltransferase assays of site-directed Bg/II linker-scanning mutations transiently transfected into primary cultures. Four positive cis-acting elements were identified and are required for efficient transcriptional activity in myogenic cells. These elements, conserved across vertebrate evolution, include the ATAAAA box (-24 bp), paired CCAAT-box-associated repeats (CBARs; at -83 bp and -127 bp), and the upstream T+A-rich regulatory sequence (at -176 bp). Basal transcriptional activity in fibroblasts was not as dependent on the upstream CBAR or regions of the upstream T+A-rich regulatory sequence. Transfection experiments provided evidence that positive regulatory factors required for alpha-actin expression in fibroblasts are limiting. In addition, negative cis-acting elements were detected and found closely associated with the G+C-rich sequences that surround the paired CBARs. Negative elements may have a role in restricting developmentally timed expression in myoblasts and appear to inhibit promoter activity in nonmyogenic cells. Cell type-specific expression of the skeletal alpha-actin gene promoter is regulated by combinatorial and possibly competitive interactions between multiple positive and negative cis-acting elements.

Glucose-induced transcription of the insulin gene is mediated by factors required for beta-cell-type-specific expression

1994 ◽  
Vol 14 (2) ◽  
pp. 871-879
Author(s):  
A Sharma ◽  
R Stein
Keyword(s):  
Beta Cell ◽  
Insulin Gene ◽  
Control Element ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Cell Extracts ◽  
Cell Type Specific Expression ◽  
Cell Type Specific

The insulin gene is expressed exclusively in pancreatic islet beta cells. The principal regulator of insulin gene transcription in the islet is the concentration of circulating glucose. Previous studies have demonstrated that transcription is regulated by the binding of trans-acting factors to specific cis-acting sequences within the 5'-flanking region of the insulin gene. To identify the cis-acting control elements within the rat insulin II gene that are responsible for regulating glucose-stimulated expression in the beta cell, we analyzed the effect of glucose on the in vivo expression of a series of transfected 5'-flanking deletion mutant constructs. We demonstrate that glucose-induced transcription of the rat insulin II gene is mediated by sequences located between -126 and -91 bp relative to the transcription start site. This region contains two cis-acting elements that are essential for directing pancreatic beta-cell-type-specific expression of the rat insulin II gene, the insulin control element (ICE; -100 to -91 bp) and RIPE3b1 (-115 to -107 bp). The gel mobility shift assay was used to determine whether the formation of the ICE- and RIPE3b1-specific factor-DNA element complexes were affected in glucose-treated beta-cell extracts. We found that RIPE3b1 binding activity was selectively induced by about eightfold. In contrast, binding to other insulin cis-acting element sequences like the ICE and RIPE3a2 (-108 to -99 bp) were unaffected by these conditions. The RIPE3b1 binding complex was shown to be distinct from the glucose-inducible factor that binds to an element located between -227 to -206 bp of the human and rat insulin I genes (D. Melloul, Y. Ben-Neriah, and E. Cerasi, Proc. Natl. Acad. Sci. USA 90:3865-3869, 1993). We have also shown that mannose, a sugar that can be metabolized by the beta cell, mimics the effects of glucose in the in vivo transfection assays and the in vitro RIPE3b1 binding assays. These results suggested that the RIPE3b1 transcription factor is a primary regulator of glucose-mediated transcription of the insulin gene. However, we found that mutations in either the ICE or the RIPE3b1 element reduced glucose-responsive expression from transfected 5'-flanking rat insulin II gene constructs. We therefore conclude that glucose-regulated transcription of the insulin gene is mediated by cis-acting elements required for beta-cell-type-specific expression.

scholarly journals Reconstitution of human Fc gamma RIII cell type specificity in transgenic mice.

1996 ◽  
Vol 183 (3) ◽  
pp. 1259-1263 ◽  
Author(s):  
M Li ◽  
U Wirthmueller ◽  
J V Ravetch
Keyword(s):  
Transgenic Mice ◽  
Nk Cells ◽  
Gene Promoter ◽  
Homologous Region ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Specific Expression ◽  
Cis Acting ◽  
Sequence Elements ◽  
Flanking Sequences

The human low affinity receptors for the Fc domain of immunoglobulin G, Fc gamma RIII, are encoded by two genes (IIIA and IIIB) which share >95% sequence identity in both coding and flanking sequences. Despite this extraordinary sequence conservation, IIIA is expressed in natural killer (NK) cells and macrophages and is absent in neutrophils, whereas IIIB is expressed only in neutrophils. To determine the molecular basis for this differential expression, we have generated transgenic mice using the genomic sequences of IIIA and IIIB. IIIA and IIIB transgenic mice show faithful reconstitution of this human pattern of cell type specificity. To determine the cis acting sequence elements that confer this specificity, we constructed chimeric genes in which 5.8 kb of 5' sequences of the IIIB gene has been replaced with a homologous region from the IIIA gene, and conversely, IIIA 5' sequences have been substituted for the analogous region of the IIIB gene. Promoter swap transgenic mice that carry IIIA 5' flanking sequences express Fc gamma RIII in macrophages and NK cells. In contrast, promoter swap transgenic mice that contain IIIB 5' sequences express Fc gamma RIII in neutrophils only. These studies define the elements conferring the cell type-specific expression of the human Fc gamma RIII genes within the 5' flanking sequences and first intron of the human Fc gamma RIIIA and Fc gamma RIIIB genes.

scholarly journals Differential repair of DNA damage in the human metallothionein gene family.

1988 ◽  
Vol 8 (12) ◽  
pp. 5331-5338 ◽  
Author(s):  
S A Leadon ◽  
M M Snowden
Keyword(s):  
Dna Damage ◽  
Gene Family ◽  
Transcriptional Activity ◽  
Initial Rate ◽  
Uv Damage ◽  
Cell Type ◽  
Specific Expression ◽  
Cell Type Specific Expression ◽  
Processed Pseudogene ◽  
Cell Type Specific

We studied the repair of UV- and aflatoxin B1 (AFB1)-induced damage in the human metallothionein (hMT) gene family. After exposure to either UV or AFB1, DNA damage was initially repaired faster in the DNA fragments containing the transcribed hMT-IA, hMT-IE, and hMT-IIA genes than in the genome overall. By 6 h posttreatment, there was at least twice as much repair in these genes as in the rest of the genome. Repair of UV damage in the hMT-IB gene, which shows cell-type specific expression, and in the hMT-IIB gene, which is a nontranscribed processed pseudogene, was about the same as in the rest of the genome, whereas repair of AFB1-induced damage was deficient in these two genes. Inducing transcription of the three expressed hMT genes with CdCl2 or of only the hMT-IIA gene with dexamethasone increased the initial rate of repair in the induced genes another twofold over the rate observed when they were transcribed at a basal level. The rates of repair in the hMT-IB and hMT-IIB genes were not altered by these inducing treatments. Transcription of the hMT genes was transiently inhibited after UV irradiation. Inducing transcription of the genes did not shorten this UV-induced delay. Thus, the efficiency of repair of damage in a DNA sequence is dependent on the level of transcriptional activity associated with that sequence. However, an increased efficiency in repair of a gene itself is not necessarily coupled to recovery of its transcription after DNA damage.

scholarly journals Pancreatic beta-cell-type-specific expression of the rat insulin II gene is controlled by positive and negative cellular transcriptional elements.

1989 ◽  
Vol 9 (8) ◽  
pp. 3253-3259 ◽  
Author(s):  
J Whelan ◽  
D Poon ◽  
P A Weil ◽  
R Stein
Keyword(s):  
Transcription Factors ◽  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Cell Type Specific Expression ◽  
Cell Type Specific

The insulin gene is expressed almost exclusively in pancreatic beta-cells. The DNA sequences that control cell-specific expression are located upstream of the transcription initiation site. To identify the cis-acting transcriptional control regions within the rat insulin II gene that are responsible for this tissue-specific expression pattern, we constructed a series of 5'-flanking deletion mutants and analyzed their expression in vivo in transfected insulin-producing and -nonproducing cell lines. Pancreatic beta-cell-specific expression was shown to be controlled by enhancer sequences lying between nucleotides -342 and -91 relative to the transcription start site. The rat insulin II enhancer appears to be a chimera, composed of a number of distinct cis-acting DNA elements. Both positive and negative transcriptional regulatory elements appear to be responsible for this cell-type-specific expression. We have shown that expression from one element within the enhancer, which is found between nucleotides -100 and -91, is regulated by both positive- and negative-acting cellular transcription factors. Expression from chimeras containing only the enhancer element sequences from -100 to -91 were active only in insulin-producing cells, indicating that the positive-acting factor(s) required for this activity may be active only in beta-cells. In contrast to the enhancer region, the rat insulin II gene promoter did not appear to require cell-specific transcription factors. Promoter mutants with 5'-flanking sequences extending to nucleotides -90 and -73 were constitutively active in both insulin-producing and -nonproducing cells. These results suggest that rat insulin II gene transcription in pancreatic beta-cells is imparted by a combination of both negative- and positive-acting cellular factors interacting with the gene enhancer.

scholarly journals The chicken skeletal alpha-actin gene promoter region exhibits partial dyad symmetry and a capacity to drive bidirectional transcription.

1988 ◽  
Vol 8 (11) ◽  
pp. 4587-4597 ◽  
Author(s):  
J M Grichnik ◽  
B A French ◽  
R J Schwartz
Keyword(s):  
Transcription Initiation ◽  
Promoter Region ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Actin Gene ◽  
High Level ◽  
Alpha Actin ◽  
Dyad Symmetry ◽  
Gene Promoter Region

The chicken skeletal alpha-actin gene promoter region (-202 to -12) provides myogenic transcriptional specificity. This promoter contains partial dyad symmetry about an axis at nucleotide -108 and in transfection experiments is capable of directing transcription in a bidirectional manner. At least three different transcription initiation start sites, oriented toward upstream sequences, were mapped 25 to 30 base pairs from TATA-like regions. The opposing transcriptional activity was potentiated upon the deletion of sequences proximal to the alpha-actin transcription start site. Thus, sequences which serve to position RNA polymerase for alpha-actin transcription may allow, in their absence, the selection of alternative and reverse-oriented start sites. Nuclear runoff transcription assays of embryonic muscle indicated that divergent transcription may occur in vivo but with rapid turnover of nuclear transcripts. Divergent transcriptional activity enabled us to define the 3' regulatory boundary of the skeletal alpha-actin promoter which retains a high level of myogenic transcriptional activity. The 3' regulatory border was detected when serial 3' deletions bisected the element (-91 CCAAA TATGG -82) which reduced transcriptional activity by 80%. Previously we showed that disruption of its upstream counterpart (-127 CCAAAGAAGG -136) resulted in about a 90% decrease in activity. These element pairs, which we describe as CCAAT box-associated repeats, are conserved in all sequenced vertebrate sarcomeric actin genes and may act in a cooperative manner to facilitate transcription in myogenic cells.

scholarly journals Glucose-induced transcription of the insulin gene is mediated by factors required for beta-cell-type-specific expression.

1994 ◽  
Vol 14 (2) ◽  
pp. 871-879 ◽  
Author(s):  
A Sharma ◽  
R Stein
Keyword(s):  
Beta Cell ◽  
Insulin Gene ◽  
Control Element ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Cell Extracts ◽  
Cell Type Specific Expression ◽  
Cell Type Specific

The insulin gene is expressed exclusively in pancreatic islet beta cells. The principal regulator of insulin gene transcription in the islet is the concentration of circulating glucose. Previous studies have demonstrated that transcription is regulated by the binding of trans-acting factors to specific cis-acting sequences within the 5'-flanking region of the insulin gene. To identify the cis-acting control elements within the rat insulin II gene that are responsible for regulating glucose-stimulated expression in the beta cell, we analyzed the effect of glucose on the in vivo expression of a series of transfected 5'-flanking deletion mutant constructs. We demonstrate that glucose-induced transcription of the rat insulin II gene is mediated by sequences located between -126 and -91 bp relative to the transcription start site. This region contains two cis-acting elements that are essential for directing pancreatic beta-cell-type-specific expression of the rat insulin II gene, the insulin control element (ICE; -100 to -91 bp) and RIPE3b1 (-115 to -107 bp). The gel mobility shift assay was used to determine whether the formation of the ICE- and RIPE3b1-specific factor-DNA element complexes were affected in glucose-treated beta-cell extracts. We found that RIPE3b1 binding activity was selectively induced by about eightfold. In contrast, binding to other insulin cis-acting element sequences like the ICE and RIPE3a2 (-108 to -99 bp) were unaffected by these conditions. The RIPE3b1 binding complex was shown to be distinct from the glucose-inducible factor that binds to an element located between -227 to -206 bp of the human and rat insulin I genes (D. Melloul, Y. Ben-Neriah, and E. Cerasi, Proc. Natl. Acad. Sci. USA 90:3865-3869, 1993). We have also shown that mannose, a sugar that can be metabolized by the beta cell, mimics the effects of glucose in the in vivo transfection assays and the in vitro RIPE3b1 binding assays. These results suggested that the RIPE3b1 transcription factor is a primary regulator of glucose-mediated transcription of the insulin gene. However, we found that mutations in either the ICE or the RIPE3b1 element reduced glucose-responsive expression from transfected 5'-flanking rat insulin II gene constructs. We therefore conclude that glucose-regulated transcription of the insulin gene is mediated by cis-acting elements required for beta-cell-type-specific expression.

Pancreatic beta-cell-type-specific expression of the rat insulin II gene is controlled by positive and negative cellular transcriptional elements

1989 ◽  
Vol 9 (8) ◽  
pp. 3253-3259
Author(s):  
J Whelan ◽  
D Poon ◽  
P A Weil ◽  
R Stein
Keyword(s):  
Transcription Factors ◽  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Cell Type ◽  
Specific Expression ◽  
Cis Acting ◽  
Cell Type Specific Expression ◽  
Cell Type Specific

The insulin gene is expressed almost exclusively in pancreatic beta-cells. The DNA sequences that control cell-specific expression are located upstream of the transcription initiation site. To identify the cis-acting transcriptional control regions within the rat insulin II gene that are responsible for this tissue-specific expression pattern, we constructed a series of 5'-flanking deletion mutants and analyzed their expression in vivo in transfected insulin-producing and -nonproducing cell lines. Pancreatic beta-cell-specific expression was shown to be controlled by enhancer sequences lying between nucleotides -342 and -91 relative to the transcription start site. The rat insulin II enhancer appears to be a chimera, composed of a number of distinct cis-acting DNA elements. Both positive and negative transcriptional regulatory elements appear to be responsible for this cell-type-specific expression. We have shown that expression from one element within the enhancer, which is found between nucleotides -100 and -91, is regulated by both positive- and negative-acting cellular transcription factors. Expression from chimeras containing only the enhancer element sequences from -100 to -91 were active only in insulin-producing cells, indicating that the positive-acting factor(s) required for this activity may be active only in beta-cells. In contrast to the enhancer region, the rat insulin II gene promoter did not appear to require cell-specific transcription factors. Promoter mutants with 5'-flanking sequences extending to nucleotides -90 and -73 were constitutively active in both insulin-producing and -nonproducing cells. These results suggest that rat insulin II gene transcription in pancreatic beta-cells is imparted by a combination of both negative- and positive-acting cellular factors interacting with the gene enhancer.

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