scholarly journals Glucocorticoid receptor-glucocorticoid response element binding stimulates nucleosome disruption by the SWI/SNF complex.

1997 ◽  
Vol 17 (2) ◽  
pp. 895-905 ◽  
Author(s):  
A K Ostlund Farrants ◽  
P Blomquist ◽  
H Kwon ◽  
O Wrange
Keyword(s):  
Glucocorticoid Receptor ◽  
Binding Site ◽  
Glucocorticoid Response Element ◽  
Response Element ◽  
Nucleosome Remodeling ◽  
Synthetic Dna ◽  
Glucocorticoid Response ◽  
Bending Sequence ◽  
Stimulation Of

The organization of DNA in chromatin is involved in repressing basal transcription of a number of inducible genes. Biochemically defined multiprotein complexes such as SWI/SNF (J. Côté, J. Quinn, J. L. Workman, and C. L. Peterson, Science 265:53-60, 1994) and nucleosome remodeling factor (T. Tsukiyama and C. Wu, Cell 83:1011-1020, 1995) disrupt nucleosomes in vitro and are thus candidates for complexes which cause chromatin decondensation during gene induction. In this study we show that the glucocorticoid receptor (GR), a hormone-inducible transcription factor, stimulates the nucleosome-disrupting activity of the SWI/SNF complex partially purified either from HeLa cells or from rat liver tissue. This GR-mediated stimulation of SWI/SNF nucleosome disruption depended on the presence of a glucocorticoid response element. The in vitro-reconstituted nucleosome probes used in these experiments harbored 95 bp of synthetic DNA-bending sequence in order to rotationally position the DNA. The GR-dependent stimulation of SWI/SNF-mediated nucleosome disruption, as evaluated by DNase I footprinting, was 2.7- to 3.8-fold for the human SWI/SNF complex and 2.5- to 3.2-fold for the rat SWI/SNF complex. When nuclear factor 1 (NF1) was used instead of GR, there was no stimulation of SWI/SNF activity in the presence of a mononucleosome containing an NF1 binding site. On the other hand, the SWI/SNF nucleosome disruption activity increased the access of NF1 for its nucleosomal binding site. No such effect was seen on binding of GR to its response element. Our results suggest that GR, but not NF1, is able to target the nucleosome-disrupting activity of the SWI/SNF complex.

scholarly journals Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription.

1989 ◽  
Vol 9 (12) ◽  
pp. 5305-5314 ◽  
Author(s):  
J Drouin ◽  
M A Trifiro ◽  
R K Plante ◽  
M Nemer ◽  
P Eriksson ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Dna Sequence ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Regulatory Element ◽  
Site Directed Mutagenesis ◽  
Glucocorticoid Response Element ◽  
Response Element ◽  
Glucocorticoid Response ◽  
Pomc Gene

Glucocorticoids rapidly and specifically inhibit transcription of the pro-opiomelanocortin (POMC) gene in the anterior pituitary, thus offering a model for studying negative control of transcription in mammals. We have defined an element within the rat POMC gene 5'-flanking region that is required for glucocorticoid inhibition of POMC gene transcription in POMC-expressing pituitary tumor cells (AtT-20). This element contains an in vitro binding site for purified glucocorticoid receptor. Site-directed mutagenesis revealed that binding of the receptor to this site located at position base pair -63 is essential for glucocorticoid repression of transcription. Although related to the well-defined glucocorticoid response element (GRE) found in glucocorticoid-inducible genes, the DNA sequence of the POMC negative glucocorticoid response element (nGRE) differs significantly from the GRE consensus; this sequence divergence may result in different receptor-DNA interactions and may account at least in part for the opposite transcriptional properties of these elements. Hormone-dependent repression of POMC gene transcription may be due to binding of the receptor over a positive regulatory element of the promoter. Thus, repression may result from mutually exclusive binding of two DNA-binding proteins to overlapping DNA sequences.

scholarly journals Identification of a functional glucocorticoid response element in the promoter of the cyclin-dependent kinase inhibitor p57Kip2

2003 ◽  
Vol 30 (3) ◽  
pp. 359-368 ◽  
Author(s):  
K Alheim ◽  
J Corness ◽  
MK Samuelsson ◽  
LG Bladh ◽  
T Murata ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Cyclin Dependent Kinase ◽  
Mobility Shift ◽  
Sequence Comparisons ◽  
Glucocorticoid Treatment ◽  
Glucocorticoid Response Element ◽  
Response Element ◽  
Glucocorticoid Response

Glucocorticoids are known regulators of the cell cycle, normally exerting an anti-proliferative effect. We have previously shown that glucocorticoids stimulate expression of p57(Kip2), a member of the Cip/Kip family of cyclin-dependent kinase inhibitors which, in some cell types, may account for the anti-proliferative responses seen after glucocorticoid treatment. The induction of p57(Kip2) involves primary transcriptional effects where no de novo protein synthesis is necessary, suggesting a direct interaction of the glucocorticoid receptor with the p57(Kip2) gene. In this study we have identified a functional glucocorticoid response element (GRE), located 5 kilo bases (kb) upstream of the transcription start site in the human p57(Kip2) promoter. This GRE was functional also when isolated, suggesting a direct transcriptional effect of the glucocorticoid receptor. Furthermore, mutation of this GRE abolished glucocorticoid induction of the reporter gene, whereas mutation of a nearby Sp1 site did not. Using electrophoretic mobility shift assays, we have shown that the -5 kb p57(Kip2) promoter GRE was able to compete with a well-known GRE for glucocorticoid receptor binding. Sequence comparisons with the mouse genome showed that this GRE is highly conserved, further strengthening the biological importance of this site. All these data emphasize the involvement of this GRE in the glucocorticoid-mediated induction of p57(Kip2) expression.

Negative regulation of transcription in vitro by a glucocorticoid response element is mediated by a trans-acting factor

1988 ◽  
Vol 8 (9) ◽  
pp. 3872-3881
Author(s):  
S J Langer ◽  
M C Ostrowski
Keyword(s):  
Mammary Tumor ◽  
Negative Regulation ◽  
Glucocorticoid Response Element ◽  
Response Element ◽  
Mammary Tumor Virus ◽  
Cell Extracts ◽  
Glucocorticoid Response ◽  
Tumor Virus ◽  
Transcription In Vitro

In vitro experiments with cell extracts prepared from a mouse mammary epithelial cell line demonstrated that a cis-acting glucocorticoid response element (GRE) of the mouse mammary tumor virus represses transcription from its homologous promoter. Competition transcription experiments, in which a molar excess of a restriction fragment that contains the GRE is added to the cell-free assay, revealed that a nuclear factor mediates in trans the negative regulation of mammary tumor virus transcription in vitro. Gel retention assays indicated that a factor in the extracts specifically recognizes the GRE. One unusual result of the gel retention studies was that heating the GRE probe to 65 degrees C before addition to a binding assay increases the formation of the specific protein-DNA complex 20-fold. Exonuclease III footprinting demonstrated that the sequences recognized by the factor are identical for either untreated or heat-treated probe. The footprinting also demonstrated that this factor recognizes sequences that are distinct from those recognized by the glucocorticoid receptor. A synthetic oligonucleotide based on the sequences identified by the footprinting experiments repressed the activity of a heterologous enhancer-promoter in vivo, as assayed by transient expression assays. We propose that this negative transcription element may control the basal level of expression of some glucocorticoid-modulated genes and may explain the insensitivity of certain tumor cells to steroid hormone action.

scholarly journals Transcriptional Analysis of the Orphan Nuclear Receptor Constitutive Androstane Receptor (NR1I3) Gene Promoter: Identification of a Distal Glucocorticoid Response Element

2003 ◽  
Vol 17 (1) ◽  
pp. 42-55 ◽  
Author(s):  
Jean Marc Pascussi ◽  
Maryvonne Busson-Le Coniat ◽  
Patrick Maurel ◽  
Marie-José Vilarem
Keyword(s):  
Glucocorticoid Receptor ◽  
Constitutive Androstane Receptor ◽  
Gene Promoter ◽  
Distal Region ◽  
Transcriptional Analysis ◽  
Pcr Analysis ◽  
Genome Database ◽  
Glucocorticoid Response Element ◽  
Response Element ◽  
Glucocorticoid Response

Abstract The constitutive androstane receptor (CAR, NR1I3) transcriptionally activates cytochrome P450 2B6, 2C9, and 3A4 when activated by xenobiotics, such as phenobarbital. Information on the human CAR promoter was obtained by searching the NCBI human genome database. A contig (NT026945) corresponding to a fragment of chromosome 1q21 was found to contain the complete CAR gene. These data were confirmed using chromosomal in situ hybridization. Both primer extension and 5′-rapid amplification of the cDNA end PCR analysis were carried out to determine the transcriptional start site of human CAR, which was found to be 32 nucleotides downstream of a potential TATA box (CATAAAA). In addition, we found that the 5′-untranslated region of CAR mRNA is 110 nucleotides shorter than previously reported. Using genomic PCR, we amplified and cloned approximately 4.9 kb (−4711/+144) of the CAR gene promoter. The activity of this promoter was measured by transient transfection. Deletion analysis suggested the presence of a glucocorticoid responsive element in its distal region (−4477/−4410). From cotransfection experiments, mutagenesis, and gel shift assays, we identified a glucocorticoid response element at −4447/−4432 that was recognized and transactivated by the human glucocorticoid receptor. Finally, using the chromatin immunoprecipitation assay, we demonstrated that the glucocorticoid receptor binds to the distal region of CAR promoter in cultured hepatocytes only in the presence of dexamethasone. Identification of this functional element provides a rational mechanistic basis for CAR induction by glucocorticoids. CAR appears to be a primary glucocorticoid receptor-response gene.

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