Identification of the Deoxyribonucleic Acid-Binding Site of a Regulatory Protein Involved in Prostate-Specific and Androgen Receptor-Dependent Gene Expression1

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

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Assessment of the Role of Activator Protein-1 on Transcription of the Mouse Steroidogenic Acute Regulatory Protein Gene

Molecular Endocrinology ◽

10.1210/me.2003-0223 ◽

2004 ◽

Vol 18 (3) ◽

pp. 558-573 ◽

Cited By ~ 52

Author(s):

Pulak R. Manna ◽

Darrell W. Eubank ◽

Douglas M. Stocco

Keyword(s):

Binding Site ◽

Gene Transcription ◽

Dna Sequences ◽

Regulatory Protein ◽

Activator Protein 1 ◽

Transcriptional Responses ◽

Activator Protein ◽

Steroidogenic Acute Regulatory ◽

Star Gene

Abstract cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even though its promoter lacks a consensus cAMP response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been demonstrated to involve combinations of DNA sequences that provide recognition motifs for sequence-specific transcription factors. We recently identified and characterized three canonical 5′-CRE half-sites within the cAMP-responsive region (−151/−1 bp) of the mouse StAR gene. Among these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1) sequence [TGA(C/G)TCA]; therefore, the role of the AP-1 transcription factor was explored in StAR gene transcription. Mutation in the AP-1 element demonstrated an approximately 50% decrease in StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequence-specific AP-1 element in StAR gene transcription was assessed using the AP-1 family members, Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to varying degrees. Disruption of the AP-1 binding site reversed the transcriptional responses seen with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further demonstrated by transfecting antisense c-Fos, Fra-1, and dominant negative forms of Fos (A-Fos) and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted in a further decrease of StAR promoter activity, demonstrating a functional cooperation between these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions between c-Fos and c-Jun with steroidogenic factor 1, GATA-4, and CCAAT/enhancer binding protein-β. These findings demonstrate that Fos and Jun can bind to the TGACTGA element in the StAR promoter and provide novel insights into the mechanisms regulating StAR gene transcription.

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Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3442 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3442-3449 ◽

Cited By ~ 16

Author(s):

M S Donoviel ◽

N Kacherovsky ◽

E T Young

Keyword(s):

Gene Expression ◽

Binding Site ◽

Reporter Gene ◽

Copy Number ◽

Glucose Repression ◽

Regulatory Protein ◽

Regulatory Region ◽

Upstream Regulatory Region ◽

Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

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The regulatory protein of glucokinase binds to the hepatocyte matrix, but, unlike glucokinase, does not translocate during substrate stimulation

Biochemical Journal ◽

10.1042/bj3090711 ◽

1995 ◽

Vol 309 (3) ◽

pp. 711-713 ◽

Cited By ~ 38

Author(s):

L Agius ◽

M Peak ◽

E Van Schaftingen

Keyword(s):

Binding Site ◽

Regulatory Protein ◽

Kinetic Properties ◽

Cell Matrix ◽

Elevated Glucose ◽

Dependent Mechanism

The kinetic properties of hepatic glucokinase (hexokinase IV) are modulated by binding to a regulatory protein. This study shows that, in hepatocytes incubated with 5 mM glucose as sole carbohydrate substrate, both glucokinase and its regulatory protein bind to the cell matrix by a Mg(2+)-dependent mechanism. After incubation with an elevated [glucose] or with fructose, glucokinase, but not its regulatory protein, translocates from the Mg(2+)-dependent binding site. It is suggested that the regulatory protein acts as a receptor for anchoring glucokinase to the hepatocyte matrix and inhibiting its activity in metabolically quiescent conditions.

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The mechanism by which the human apolipoprotein B gene reducer operates involves blocking of transcriptional activation by hepatocyte nuclear factor 3.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1534 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1534-1546 ◽

Cited By ~ 36

Author(s):

B Paulweber ◽

F Sandhofer ◽

B Levy-Wilson

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Apolipoprotein B ◽

Regulatory Protein ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Apo B ◽

Human Apolipoprotein ◽

Colon Carcinoma Cells ◽

Mediator Protein

Previously, we showed that when a DNA fragment extending from -3067 to -2734 of the human apolipoprotein B (apo-B) gene is inserted immediately upstream of an apo-B promoter segment (-139 to +121), transcription from this promoter is reduced by about 10-fold in cultured colon carcinoma cells (CaCo-2) but not in cultured hepatoma cells (HepG2). We postulated that this reducer operates by a mechanism involving active repression of a transcriptional activator that binds to the segment from -111 to -88 of the apo-B promoter (B. Paulweber and B. Levy-Wilson, J. Biol. Chem. 266:24161-24168 1991). In the current study, the reducer element has been localized to a 24-bp sequence from -2801 to -2778 of the apo-B gene that contains a binding site for the negative regulatory protein ARP-1. Furthermore, we have demonstrated that the transcription factor hepatocyte nuclear factor 3 alpha (HNF-3 alpha) binds to the sequence 5'-TGTTTGCTTTTC-3' from -95 to -106 of the apo-B promoter, to stimulate transcription. Transcriptional activation by HNF-3 is repressed when the reducer sequence is inserted immediately upstream of the HNF-3 binding site, suggesting a mechanism by which the reducer-bound protein blocks the activation promoted by HNF-3. Data from cotransfection experiments in which ARP-1 is overexpressed in the absence of its binding site suggest that ARP-1 interacts either directly or via a mediator protein with proteins recognizing the HNF-3 site and that this interaction is sufficient to repress transcriptional activation by HNF-3. Because transcriptional activation by Sp1 is not affected by the reducer, it is unlikely that the reducer interacts directly with basic components of the transcriptional machinery.

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