A transcriptionally active DNA-binding site for human p53 protein complexes.

Recent studies have demonstrated transcriptional activation domains within the tumor suppressor protein p53, while others have described specific DNA-binding sites for p53, implying that the protein may act as a transcriptional regulatory factor. We have used a reiterative selection procedure (CASTing: cyclic amplification and selection of targets) to identify new specific binding sites for p53, using nuclear extracts from normal human fibroblasts as the source of p53 protein. The preferred consensus is the palindrome GGACATGCCCGGGCATGTCC. In vitro-translated p53 binds to this sequence only when mixed with nuclear extracts, suggesting that p53 may bind DNA after posttranslational modification or as a complex with other protein partners. When placed upstream of a reporter construct, this sequence promotes p53-dependent transcription in transient transfection assays.

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Transcriptional control of the factor IX gene: analysis of five cis- acting elements and the deleterious effects of naturally occurring hemophilia B Leyden mutations

Blood ◽

10.1182/blood.v84.9.2992.2992 ◽

1994 ◽

Vol 84 (9) ◽

pp. 2992-3000 ◽

Cited By ~ 22

Author(s):

DJ Picketts ◽

CR Mueller ◽

D Lillicrap

Keyword(s):

Dna Binding ◽

Protein Binding ◽

Transcriptional Activation ◽

In Vitro Transcription ◽

Factor Ix ◽

Hemophilia B ◽

Transcription Assay ◽

Cis Acting ◽

Nuclear Extracts

Abstract Hemophilia B Leyden is a rare form of inherited factor IX deficiency in which patients experience spontaneous postpubertal recovery of factor IX levels. The mutations resulting in this disorder are localized in a 40-nucleotide region encompassing the major transcriptional start site for factor IX. Here we report the further characterization of five cis- acting elements in the factor IX promoter and the effects on protein binding and transcriptional activation of five Leyden mutations (at nucleotides +13, -5, -6, -20, and -26) that occur within the proximal three elements (sites 1 through 3). Bandshift studies using nuclear extracts from four different rat tissues have shown that at least some of the proteins binding to each of the five sites are ubiquitous in nature. The pattern of DNA binding at site 1 suggests that this element plays an important role in mediating the liver-specific expression of factor IX. Additional studies with liver nuclear extracts obtained at several different points in development have shown an increase in DNA binding at sites 1, 4, and 5 between 1 day and 1 week. Using DNase I footprint analysis and competition bandshift studies, we have shown that the binding of nuclear proteins to each of the mutant sites is disrupted to a variable extent. There appears to be some, although reduced, protein binding to all of the mutant oligonucleotides apart from the -26 mutant. In vitro transcription assays have shown that each of the mutations reduces the global proximal promoter activity by approximately 40%. Two double mutant promoters did not show any additional downregulation in the in vitro transcription assay. In experiments designed to assess the relative transcriptional activity mediated from each of the five sites independently, we have tested artificial homopolymer promoters of each site in the in vitro transcription assay. These studies show that sites 4 and 5 are the strongest activators and that transactivation from site 5 is further enhanced by the albumin D site-binding protein. In summary, these investigations show deleterious effects of each of the Leyden mutations tested on the binding of trans-acting factors and also show disruption of transcriptional activation in a functional in vitro transcription assay. Our results also show that cis-acting elements 4 and 5 are the principal activators of this locus.

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Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1405 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1405-1421 ◽

Cited By ~ 197

Author(s):

C C Adams ◽

J L Workman

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Sites ◽

Specific Binding ◽

General Mechanism ◽

Nf Kappa B ◽

Nucleosomal Dna ◽

Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

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A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.887 ◽

1990 ◽

Vol 10 (3) ◽

pp. 887-897 ◽

Cited By ~ 72

Author(s):

A R Buchman ◽

R D Kornberg

Keyword(s):

Dna Sequences ◽

Transcriptional Activation ◽

Binding Sites ◽

Essential Gene ◽

Specific Binding ◽

Binding Protein ◽

Point Mutations ◽

Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

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Drosophila Ada2b Is Required for Viability and Normal Histone H3 Acetylation

Molecular and Cellular Biology ◽

10.1128/mcb.24.18.8080-8089.2004 ◽

2004 ◽

Vol 24 (18) ◽

pp. 8080-8089 ◽

Cited By ~ 37

Author(s):

Dai Qi ◽

Jan Larsson ◽

Mattias Mannervik

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Protein Complexes ◽

P53 Protein ◽

Polytene Chromosomes ◽

Histone H3 ◽

Saga Complex ◽

Histone H3 Acetylation ◽

H3 Acetylation

ABSTRACT Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent.

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An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4174-4185.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4174-4185

Author(s):

R Buettner ◽

P Kannan ◽

A Imhof ◽

R Bauer ◽

S O Yim ◽

...

Keyword(s):

Retinoic Acid ◽

Dna Binding ◽

Transcriptional Activation ◽

Specific Binding ◽

Soft Agar ◽

Negative Regulator ◽

Dimerization Domain ◽

Teratocarcinoma Cell ◽

Nuclear Extracts ◽

Alternatively Spliced

AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spanning the entire AP-2 gene proves that AP-2A and AP-2B transcripts are alternatively spliced from the same gene. Both transient and stable transfection experiments show that AP-2B inhibits AP-2 transactivator function, as measured by an AP-2-responsive chloramphenicol acetyltransferase reporter plasmid. Furthermore, constitutive AP-2B expression in PA-1 cells causes a retinoic acid-resistant phenotype, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, in a fashion similar to transformation of these cells by oncogenes. To determine the mechanism by which AP-2B exerts its inhibitory function, we purified bacterially expressed AP-2A and AP-2B proteins. While bacterial AP-2B does not bind an AP-2 consensus site, it strongly inhibits binding of the endogenous AP-2 present in PA-1 cell nuclear extracts. However, DNA sequence-specific binding of bacterially expressed AP-2A cannot be inhibited by bacterially expressed AP-2B. Therefore, inhibition of AP-2 activity by the protein AP-2B may require an additional factor or modification supplied by nuclear extracts.

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Molecular Characterization of a PU.1 Transcription Complex Formed on the IL-1β Proximal Promoter.

Blood ◽

10.1182/blood.v104.11.3547.3547 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3547-3547

Author(s):

Trang Hoang ◽

Benoit Grondin ◽

Martin Lefrancois ◽

Marianne St Denis ◽

Daniel G. Tenen ◽

...

Keyword(s):

Gene Expression ◽

Stress Response ◽

Dna Binding ◽

Leucine Zipper ◽

Protein Complexes ◽

Proximal Promoter ◽

Stress Exposure ◽

Protein Protein Interaction ◽

Nuclear Extracts

Abstract The gene coding for the pro-inflammatory cytokine IL-1β is induced at the transcription level in differentiating macrophages and in stress response. Interestingly, PU.1 and C/EBPβ, two transcription factors implicated in IL-1β gene expression are not induced by stress exposure, while c-Jun is strongly induced. Strikingly, this upregulation of c-Jun is required for IL-1β induction, as cells expressing a c-Jun antisense construct fail to respond to stress exposure. We have mapped the induction of IL-1β gene expression to its proximal promoter and show that it is mediated by the transcriptional synergy between C/EBPβ, c-Jun and PU.1 via specific DNA binding sites for C/EBPβ and PU.1 only. To elucidate how PU.1 and C/EBPβ cooperate with c-Jun at the molecular level, we have optimized a DNA binding assay based on IL-1β promoter fragments immobilized on beads to isolate protein complexes from nuclear extracts, which were subsequently eluted and identified by Western blotting. We show that PU.1 or C/EBPβ alone directly bind this promoter fragment via specific sequences while purified recombinant c-Jun fails to do so. However, the presence of either PU.1 or C/EBPβ on the promoter allows for a recruitment of c-Jun to the DNA template, mediated by direct protein-protein interaction. Interestingly, the leucine zipper domain of c-Jun is essential for its interaction with C/EBPβ while dispensable for PU.1 interaction in vitro whereas its basic domain is required for both interactions. Furthermore, we show that PU.1 and C/EBPβ cooperatively bind the IL-1β promoter, resulting in a synergistic recruitment of c-Jun. Finally, we show that the strength of interaction of c-Jun mutants with PU.1 or C/EBPβ determine the strength of transcription output and c-Jun mutants that fail to associate with either PU.1 or C/EBPβ are transcriptionally inactive. In contrast, c-Jun mutants exhibiting increased homodimerization are more active that the wild type protein. Taken together, our data suggest that c-Jun homodimers can be targeted to the IL-1β promoter in the absence of a specific DNA binding element, and conclude that PU.1 and C/EBPβ are specifically tethered to the IL-1β promoter while c-Jun cooperatively binds these proteins and acts as a transcriptional co-activator. We propose a mechanism based on an initial binding of PU.1 and C/EBPβ to the IL-1β promoter followed by a cooperative recruitment of c-Jun, resulting in transcriptional synergy and IL-1β gene expression in stress response.

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Activator Protein-2 Mediates Transcriptional Activation of the CYP11A1 Gene by Interaction with Sp1 Rather than Binding to DNA

Molecular Endocrinology ◽

10.1210/mend.13.8.0335 ◽

1999 ◽

Vol 13 (8) ◽

pp. 1402-1416 ◽

Cited By ~ 18

Author(s):

Pilar Pena ◽

Anne T. Reutens ◽

Chris Albanese ◽

Mark D’Amico ◽

Genichi Watanabe ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Sites ◽

Luciferase Reporter ◽

Carboxy Terminus ◽

Luciferase Reporter Gene ◽

Activator Protein ◽

Basic Region ◽

Activator Protein 2

Abstract The ovine P450 side chain cleavage (CYP11A1) enzyme gene, which catalyzes the initial enzymatic step in steroid hormone biosynthesis is transcriptionally regulated in cultured steroidogenic human trophoblastic JEG-3 cells. The ovine CYP11A1 promoter contains two GC-rich footprinted regions referred to as ovine footprints 5 (OF5) and OF3, which are well conserved among the CYP11A1 promoters of different species. These GC-rich sequences resemble activator protein-2 (AP-2)/Sp1 binding sites and were previously implicated in basal and cAMP-regulated activity of the bovine and ovine CYP11A1 promoters. In the current studies, AP-2 induced the ovine CYP11A1 promoter 4.5-fold in JEG-3 cells with full induction requiring the previously defined cAMP-responsive elements. Point mutation of OF3 abolished induction by AP-2, and OF3 was sufficient for induction by AP-2 when linked to a heterologous promoter. AP-2 induction of the CYP11A1 promoter required the basic region (N165-N278) and the carboxy terminus of AP-2 (N413-N437). In the course of investigating the mechanisms by which OF5 and OF3 regulated CYP11A1 transcription, we found that OF5 and OF3 bound Sp1 and Sp3 in JEG-3 cells. AP-2 did not bind OF5 or OF3 directly but rather formed a multiprotein complex with Sp1 in JEG-3 cells. AP-2 associated directly with Sp1 in vitro requiring the AP-2 basic region and the Sp1 carboxy terminus. AP-2 induced Sp1/Sp3 activity independently of AP-2 binding to DNA using a GAL4 paradigm. The Sp1 and Sp3 transactivation domains were linked to the DNA-binding domain of GAL4, and their activity was assessed using a luciferase reporter gene containing only the GAL4 DNA-binding sites linked to the minimal TATA site. AP-2 induced Sp1/Sp3-GAL4 activity 3- to 4-fold, requiring both the amino and extreme carboxy terminus of AP-2. We conclude that AP-2 can bind to and stimulate Sp1 activity and induces the ovine CYP11A1 promoter through conserved Sp1/Sp3-binding sites in JEG-3 cells. The induction of Sp1 activity by AP-2 may contribute to the induction of other genes that bind Sp1.

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An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4174 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4174-4185 ◽

Cited By ~ 87

Author(s):

R Buettner ◽

P Kannan ◽

A Imhof ◽

R Bauer ◽

S O Yim ◽

...

Keyword(s):

Retinoic Acid ◽

Dna Binding ◽

Transcriptional Activation ◽

Specific Binding ◽

Soft Agar ◽

Negative Regulator ◽

Dimerization Domain ◽

Teratocarcinoma Cell ◽

Nuclear Extracts ◽

Alternatively Spliced

AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spanning the entire AP-2 gene proves that AP-2A and AP-2B transcripts are alternatively spliced from the same gene. Both transient and stable transfection experiments show that AP-2B inhibits AP-2 transactivator function, as measured by an AP-2-responsive chloramphenicol acetyltransferase reporter plasmid. Furthermore, constitutive AP-2B expression in PA-1 cells causes a retinoic acid-resistant phenotype, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, in a fashion similar to transformation of these cells by oncogenes. To determine the mechanism by which AP-2B exerts its inhibitory function, we purified bacterially expressed AP-2A and AP-2B proteins. While bacterial AP-2B does not bind an AP-2 consensus site, it strongly inhibits binding of the endogenous AP-2 present in PA-1 cell nuclear extracts. However, DNA sequence-specific binding of bacterially expressed AP-2A cannot be inhibited by bacterially expressed AP-2B. Therefore, inhibition of AP-2 activity by the protein AP-2B may require an additional factor or modification supplied by nuclear extracts.

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A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.887-897.1990 ◽

1990 ◽

Vol 10 (3) ◽

pp. 887-897

Author(s):

A R Buchman ◽

R D Kornberg

Keyword(s):

Dna Sequences ◽

Transcriptional Activation ◽

Binding Sites ◽

Essential Gene ◽

Specific Binding ◽

Binding Protein ◽

Point Mutations ◽

Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

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