scholarly journals E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

1990 ◽  
Vol 10 (10) ◽  
pp. 5138-5149 ◽  
Author(s):  
R J Rooney ◽  
P Raychaudhuri ◽  
J R Nevins
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Stable Complex ◽  
Small Subset ◽  
Adenovirus Infection ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Sequence Recognition ◽  
Nuclear Factors ◽  
Dna Binding Factors

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation

1990 ◽  
Vol 10 (10) ◽  
pp. 5138-5149
Author(s):  
R J Rooney ◽  
P Raychaudhuri ◽  
J R Nevins
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Stable Complex ◽  
Small Subset ◽  
Adenovirus Infection ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Sequence Recognition ◽  
Nuclear Factors ◽  
Dna Binding Factors

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

scholarly journals Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation.

1994 ◽  
Vol 14 (12) ◽  
pp. 8420-8431 ◽  
Author(s):  
M Xu ◽  
K A Sheppard ◽  
C Y Peng ◽  
A S Yee ◽  
H Piwnica-Worms
Keyword(s):  
Dna Binding ◽  
Active Form ◽  
Cyclin A ◽  
Binding Activity ◽  
Stable Complex ◽  
Binding Studies ◽  
Dna Binding Activity ◽  
Phosphopeptide Mapping

E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G1-to-S phase transition in higher eukaryotic cells. E2F-1 forms a heterodimer with DP-1 and binds to several cell cycle regulatory proteins, including the retinoblastoma family (RB, p107, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphorylation and its interaction with cyclin A/CDK2 complexes both in vivo and in vitro. In vitro, E2F-1 formed a stable complex with cyclin A/CDK2 but not with either subunit alone. DP-1 did not interact with cyclin A, CDK2, or the cyclin A/CDK2 complex. While the complex of cyclin A/CDK2 was required for stable complex formation with E2F-1, the kinase-active form of CDK2 was not required. However, E2F-1 was phosphorylated by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa cells. Two-dimensional tryptic phosphopeptide mapping studies demonstrated an overlap in the phosphopeptides derived from E2F-1 labeled in vitro and in vivo, indicating that cyclin A/CDK2 may be responsible for the majority of E2F-1 phosphorylation in vivo. Furthermore, an active DNA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo demonstrated that the cyclin A/CDK2-binding region resided within the N-terminal 124 amino acids of E2F-1. Because the stable association of E2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- or RB-binding domain and because the interactions could be reconstituted from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that the DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation by cyclin A/CDK2.

cAMP response element-binding protein phosphorylation and DNA binding activity are increased in the anterior pituitary gland following glucocorticoid depletion

1997 ◽  
Vol 19 (3) ◽  
pp. 291-297 ◽  
Author(s):  
D Whitehead ◽  
DA Carter
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Camp Response Element Binding ◽  
Binding Activity ◽  
Binding Assays ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Protein ◽  
Dna Binding Activity ◽  
Element Binding Protein

Activation of the hypothalamo-pituitary-adrenal (HPA) axis during stress is associated with increased expression of genes that code for regulatory hormones such as corticotrophin-releasing factor (CRF) and ACTH. The identity of the transcription factors that mediate these changes in gene expression is not known. In the present study we have investigated the expression of the cAMP response-element binding protein (CREB) in mouse pituitary, and its regulation during a pharmacological paradigm that simulates activation of the CRF-ACTH axis. Using Western blots and DNA binding assays we have shown that both CREB protein (43 kDa) and CRE binding exhibit a readily-detectable basal level of activity in the pituitary. Following treatment with the 11 beta-hydroxylase inhibitor metyrapone, CRE binding activity was increased at 1 and 2 h but levels of CREB protein were not found to be consistently elevated. However, using a Ser133 phosphopeptide-specific antibody, that detects the functionally important phosphorylated form of CREB (P-CREB), we have shown that levels of pituitary P-CREB are markedly elevated following metyrapone. The same antibody was also used in DNA binding assays, and in the presence of this antiserum CRE binding activity in samples extracted from metyrapone-treated animals was reduced to levels similar to controls. Parallel experiments have confirmed previous studies showing increases in c-Fos expression and AP-1 DNA binding activity following metyrapone treatment but we have shown that c-Fos-associated binding activity does not appear to contribute to the increase in activity detected using the CRE binding probe. Our evidence of functionally relevant changes in pituitary CREB activity following glucocorticoid depletion must be viewed in the context of numerous other novel pituitary transcription factors that are implicated in HPA regulation, but our use of mice as an experimental model has facilitated the use of novel mouse mutants that can be used to dissect the role of individual factors.

scholarly journals Strong binding activity of few transcription factors is a major determinant of open chromatin

2017 ◽  
Author(s):  
Bei Wei ◽  
Arttu Jolma ◽  
Biswajyoti Sahu ◽  
Lukas M. Orre ◽  
Fan Zhong ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Large Fraction ◽  
Cell Types ◽  
Binding Activity ◽  
Tissue Type ◽  
Small Subset ◽  
Open Chromatin ◽  
Protein Activity ◽  
Dna Binding Activity

AbstractIt is well established that transcription factors (TFs) play crucial roles in determining cell identity, and that a large fraction of all TFs are expressed in most cell types. In order to globally characterize activities of TFs in cells, we have developed a novel massively parallel protein activity assay, Active TF Identification (ATI) that measures DNA-binding activity of all TFs from any species or tissue type. In contrast to previous studies based on mRNA expression or protein abundance, we found that a set of TFs binding to only around ten distinct motifs display strong DNA-binding activity in any given cell or tissue type. Mass spectrometric identification of TFs revealed that within these highly active TFs, there were both housekeeping TFs, which were universally found in all cell types, and specific TFs, which were highly enriched in known factors that determine the fate of the analyzed tissue or cell type. The importance of a small subset of TFs for determining the overall accessible chromatin landscape of a cell suggests that gene regulatory logic may be simpler than what has previously been appreciated.

Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation

1994 ◽  
Vol 14 (12) ◽  
pp. 8420-8431
Author(s):  
M Xu ◽  
K A Sheppard ◽  
C Y Peng ◽  
A S Yee ◽  
H Piwnica-Worms
Keyword(s):  
Dna Binding ◽  
Active Form ◽  
Cyclin A ◽  
Binding Activity ◽  
Stable Complex ◽  
Binding Studies ◽  
Dna Binding Activity ◽  
Phosphopeptide Mapping

E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G1-to-S phase transition in higher eukaryotic cells. E2F-1 forms a heterodimer with DP-1 and binds to several cell cycle regulatory proteins, including the retinoblastoma family (RB, p107, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphorylation and its interaction with cyclin A/CDK2 complexes both in vivo and in vitro. In vitro, E2F-1 formed a stable complex with cyclin A/CDK2 but not with either subunit alone. DP-1 did not interact with cyclin A, CDK2, or the cyclin A/CDK2 complex. While the complex of cyclin A/CDK2 was required for stable complex formation with E2F-1, the kinase-active form of CDK2 was not required. However, E2F-1 was phosphorylated by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa cells. Two-dimensional tryptic phosphopeptide mapping studies demonstrated an overlap in the phosphopeptides derived from E2F-1 labeled in vitro and in vivo, indicating that cyclin A/CDK2 may be responsible for the majority of E2F-1 phosphorylation in vivo. Furthermore, an active DNA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo demonstrated that the cyclin A/CDK2-binding region resided within the N-terminal 124 amino acids of E2F-1. Because the stable association of E2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- or RB-binding domain and because the interactions could be reconstituted from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that the DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation by cyclin A/CDK2.

scholarly journals A novel allele of HAP1 causes uninducible expression of HEM13 in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 136 (3) ◽  
pp. 819-831 ◽  
Author(s):  
S C Ushinsky ◽  
T Keng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Mutant Allele ◽  
Binding Activity ◽  
The Other ◽  
Wild Type ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Allele Expression ◽  
Novel Allele

Abstract Transcription of HEM13 in Saccharomyces cerevisiae is repressed by heme and oxygen. We have isolated two mutants in which expression of HEM13 is aberrant. The mutant alleles in these strains represent two different alleles of HAP1. HAP1 encodes an activator protein whose DNA binding activity is stimulated by heme, and is required for the transcription of CYC1, ROX1 and a number of other heme-dependent genes. One of our mutant alleles confers a phenotype much like that of the hap1::LEU2 allele. Expression of HEM13 in a strain with this mutation is elevated under repressing conditions and not fully inducible in the absence of heme. The other mutant allele of HAP1 we uncovered confers a novel phenotype. A strain containing this allele exhibits heme-independent expression of CYC1 and ROX1 and uninducible expression of HEM13 and ANB1. The mutation associated with this novel allele of HAP1 was localized to a glycine to aspartate change in amino acid 235 of HAP1, between the DNA binding and heme responsive domains. DNA binding assays demonstrated that the protein made from this HAP1 allele retains the ability to bind DNA, but that unlike wild-type HAP1 protein, this binding is not stimulated by heme.

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