scholarly journals Identification of an estrogen-responsive element from the 5'-flanking region of the rat prolactin gene.

1987 ◽  
Vol 7 (12) ◽  
pp. 4247-4254 ◽  
Author(s):  
R A Maurer ◽  
A C Notides
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Intrinsic Property ◽  
Conserved Sequence ◽  
Prolactin Gene ◽  
Flanking Region ◽  
Estrogenic Regulation

The DNA sequences which interact with the estrogen receptor and which mediate the estrogenic regulation of prolactin gene transcription have been investigated by the use of receptor-DNA-binding experiments and gene transfer studies. Nitrocellulose filter binding assays using highly purified estrogen receptor and cloned fragments of the 5'-flanking region of the rat prolactin gene demonstrate that the receptor selectively binds to DNA sequences located between nucleotides -1713 and -1532 with respect to the transcription initiation site. The binding of the estrogen receptor to this region of the prolactin gene was strongly dependent on receptor concentration, suggesting that receptor dimers may be important in DNA binding. These data demonstrate that the selective binding of purified estrogen receptor to specific sequences of the rat prolactin gene is an intrinsic property of the receptor and is not due to the interaction of receptor with other proteins. The role of specific prolactin gene sequences in mediating the estrogenic regulation of prolactin gene transcription was confirmed by the use of prolactin-chloramphenicol acetyltransferase fusion genes. These studies demonstrated that sequences upstream of position -1532 are required for estrogen responsiveness. Furthermore, the region of the prolactin gene at -1713 to -1495 was able to confer estrogen responsiveness on the thymidine kinase promoter. Exonuclease III protection experiments further localized the receptor-binding sequences to positions -1587 to -1563. Comparison of the nucleotide sequence of the region of the prolactin gene which binds the estrogen receptor with the sequence of other estrogen-responsive genes suggested the presence of the conserved sequence [sequence in text], which shows similarity to sequences thought to mediate glucocorticoid receptor effects on transcription.

Identification of an estrogen-responsive element from the 5'-flanking region of the rat prolactin gene

1987 ◽  
Vol 7 (12) ◽  
pp. 4247-4254
Author(s):  
R A Maurer ◽  
A C Notides
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Intrinsic Property ◽  
Conserved Sequence ◽  
Prolactin Gene ◽  
Flanking Region ◽  
Estrogenic Regulation

The DNA sequences which interact with the estrogen receptor and which mediate the estrogenic regulation of prolactin gene transcription have been investigated by the use of receptor-DNA-binding experiments and gene transfer studies. Nitrocellulose filter binding assays using highly purified estrogen receptor and cloned fragments of the 5'-flanking region of the rat prolactin gene demonstrate that the receptor selectively binds to DNA sequences located between nucleotides -1713 and -1532 with respect to the transcription initiation site. The binding of the estrogen receptor to this region of the prolactin gene was strongly dependent on receptor concentration, suggesting that receptor dimers may be important in DNA binding. These data demonstrate that the selective binding of purified estrogen receptor to specific sequences of the rat prolactin gene is an intrinsic property of the receptor and is not due to the interaction of receptor with other proteins. The role of specific prolactin gene sequences in mediating the estrogenic regulation of prolactin gene transcription was confirmed by the use of prolactin-chloramphenicol acetyltransferase fusion genes. These studies demonstrated that sequences upstream of position -1532 are required for estrogen responsiveness. Furthermore, the region of the prolactin gene at -1713 to -1495 was able to confer estrogen responsiveness on the thymidine kinase promoter. Exonuclease III protection experiments further localized the receptor-binding sequences to positions -1587 to -1563. Comparison of the nucleotide sequence of the region of the prolactin gene which binds the estrogen receptor with the sequence of other estrogen-responsive genes suggested the presence of the conserved sequence [sequence in text], which shows similarity to sequences thought to mediate glucocorticoid receptor effects on transcription.

scholarly journals Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

1985 ◽  
Vol 5 (11) ◽  
pp. 2967-2974 ◽  
Author(s):  
B A White ◽  
G M Preston ◽  
T C Lufkin ◽  
C Bancroft
Keyword(s):  
Transcription Initiation ◽  
Genomic Clone ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Sequence Specificity ◽  
Strong Binding ◽  
Prolactin Gene ◽  
Rat Pituitary ◽  
Chromatin Proteins ◽  
Flanking Region

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

scholarly journals Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

1991 ◽  
Vol 11 (6) ◽  
pp. 2937-2945 ◽  
Author(s):  
E Martinez ◽  
Y Dusserre ◽  
W Wahli ◽  
N Mermod
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Limiting Factor ◽  
Gene Promoters ◽  
Protein Coding ◽  
Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene

1985 ◽  
Vol 5 (11) ◽  
pp. 2967-2974
Author(s):  
B A White ◽  
G M Preston ◽  
T C Lufkin ◽  
C Bancroft
Keyword(s):  
Transcription Initiation ◽  
Genomic Clone ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Sequence Specificity ◽  
Strong Binding ◽  
Prolactin Gene ◽  
Rat Pituitary ◽  
Chromatin Proteins ◽  
Flanking Region

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

scholarly journals Domain analysis of the plant DNA-binding protein GT1a: requirement of four putative alpha-helices for DNA binding and identification of a novel oligomerization region.

1995 ◽  
Vol 15 (2) ◽  
pp. 1014-1020 ◽  
Author(s):  
E Lam
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Cdna Clones ◽  
Protein Oligomerization ◽  
Gene Promoters ◽  
Alpha Helices

Light is an important environmental signal that can influence diverse developmental processes in plants. Many plant nuclear genes respond to light at the level of transcription initiation. GT-1 and GT2 are nuclear factors which interact with DNA sequences in many light-responsive gene promoters. cDNA clones which encode proteins with sequence binding specificities similar to those of these two factors have been isolated. They show significant amino acid sequence similarities within three closely spaced, putative alpha-helices that were predicted by secondary structure analysis but do not show significant homologies with any other reported DNA-binding protein. In this work, N- and C-terminal deletions of tobacco GT1a were generated by in vitro transcription and translation, and their DNA-binding activities and subunit structures were studied. The results suggest that the C-terminal domain of GT1a is critical for protein oligomerization, while a region predicted to contain four closely spaced alpha-helices is required for DNA binding. Direct chemical cross-linking and gel filtration analyses of full-length and truncated derivatives of GT1a suggest that this factor can exist in solution as a homotetramer and that oligomerization is independent of DNA binding. This study thus establishes two independent functional domains in this class of eukaryotic trans-acting factors. Possible implications of the multimeric nature of GT1a in relation to the known characteristics of light-responsive promoter architecture are discussed.

Insulin gene enhancer activity is inhibited by adenovirus 5 E1a gene products

1989 ◽  
Vol 9 (10) ◽  
pp. 4531-4534
Author(s):  
R W Stein ◽  
J Whelan
Keyword(s):  
Transcription Factors ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Insulin Gene ◽  
Target Sequence ◽  
Flanking Region ◽  
Cellular Transcription ◽  
Insulin Gene Transcription

Selective transcription of the insulin gene in pancreatic beta cells is regulated by its enhancer, located within the 5'-flanking region of the insulin gene. Transcription from the enhancer is controlled by both positive- and negative-acting cellular transcription factors. It was previously shown that both the 243- and 289-amino-acid adenovirus type 5 E1a proteins can repress insulin gene transcription in vivo. To localize the insulin DNA sequences involved in this response, we examined the effects of a number of mutations within the 5'-flanking region of the rat insulin II gene on E1a-mediated repression of insulin gene transcription. We have found that E1a proteins inhibit enhancer-stimulated transcription of the insulin gene. The enhancer appears to contain at least two genetically separable and independent E1a target sequence elements. Interestingly, these same regions of the insulin enhancer have been shown to be negatively regulated by cellular transcription factors. These results suggest that E1a-like cellular factors may function in the pancreatic beta-cell-specific expression of the insulin gene.

scholarly journals Insulin gene enhancer activity is inhibited by adenovirus 5 E1a gene products.

1989 ◽  
Vol 9 (10) ◽  
pp. 4531-4534 ◽  
Author(s):  
R W Stein ◽  
J Whelan
Keyword(s):  
Transcription Factors ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Insulin Gene ◽  
Target Sequence ◽  
Flanking Region ◽  
Cellular Transcription ◽  
Insulin Gene Transcription

Selective transcription of the insulin gene in pancreatic beta cells is regulated by its enhancer, located within the 5'-flanking region of the insulin gene. Transcription from the enhancer is controlled by both positive- and negative-acting cellular transcription factors. It was previously shown that both the 243- and 289-amino-acid adenovirus type 5 E1a proteins can repress insulin gene transcription in vivo. To localize the insulin DNA sequences involved in this response, we examined the effects of a number of mutations within the 5'-flanking region of the rat insulin II gene on E1a-mediated repression of insulin gene transcription. We have found that E1a proteins inhibit enhancer-stimulated transcription of the insulin gene. The enhancer appears to contain at least two genetically separable and independent E1a target sequence elements. Interestingly, these same regions of the insulin enhancer have been shown to be negatively regulated by cellular transcription factors. These results suggest that E1a-like cellular factors may function in the pancreatic beta-cell-specific expression of the insulin gene.

Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor

1991 ◽  
Vol 11 (6) ◽  
pp. 2937-2945
Author(s):  
E Martinez ◽  
Y Dusserre ◽  
W Wahli ◽  
N Mermod
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Limiting Factor ◽  
Gene Promoters ◽  
Protein Coding ◽  
Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

scholarly journals Structural basis of DNA binding to human YB-1 cold shock domain regulated by phosphorylation

2020 ◽  
Vol 48 (16) ◽  
pp. 9361-9371 ◽  
Author(s):  
Jingfeng Zhang ◽  
Jing-Song Fan ◽  
Shuangli Li ◽  
Yunhuang Yang ◽  
Peng Sun ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Cold Shock ◽  
Structural Basis ◽  
Nucleic Acid Metabolism ◽  
Multifunctional Protein ◽  
Dna And Rna ◽  
Cold Shock Domain

Abstract Human Y-box binding protein 1 (YB-1) is a multifunctional protein and overexpressed in many types of cancer. It specifically recognizes DNA/RNA through a cold shock domain (CSD) and regulates nucleic acid metabolism. The C-terminal extension of CSD and the phosphorylation of S102 are indispensable for YB-1 function. Until now, the roles of the C-terminal extension and phosphorylation in gene transcription and translation are still largely unknown. Here, we solved the structure of human YB-1 CSD with a C-terminal extension sequence (CSDex). The structure reveals that the extension interacts with several residues in the conventional CSD and adopts a rigid structure instead of being disordered. Either deletion of this extension or phosphorylation of S102 destabilizes the protein and results in partial unfolding. Structural characterization of CSDex in complex with a ssDNA heptamer shows that all the seven nucleotides are involved in DNA–protein interactions and the C-terminal extension provides a unique DNA binding site. Our DNA-binding study indicates that CSDex can recognize more DNA sequences than previously thought and the phosphorylation reduces its binding to ssDNA dramatically. Our results suggest that gene transcription and translation can be regulated by changing the affinity of CSDex binding to DNA and RNA through phosphorylation, respectively.

Sign in / Sign up

Export Citation Format

Share Document