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O Wrange

Publications and source records attributed to O Wrange.

At least 19 recordsLinked to original sources
PubMed2001

Hormone-induced nucleosome positioning in the MMTV promoter is reversible.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone via the glucocorticoid receptor (GR). The hormone-triggered effects on MMTV transcription and chromatin structure were studied in Xenopus oocytes. We previously showed that the nucleosomes organizing the MMTV promoter became translationally positioned upon hormone induction. A single GR-binding site was necessary and sufficient for the chromatin events to occur, while transcription and basal promoter elements were dispensable. Here we show that addition of the hormone antagonists RU486 or RU43044 to the previously hormone-induced MMTV promoter results in cessation of transcription and loss of chromatin remodeling and nucleosome positioning. In vivo footprinting demonstrated agonist- and RU486-induced GR binding to its DNA response element (GRE), while the other antagonist, RU43044, did not promote GR-GRE interaction. These results demonstrate that induction and maintenance of nucleosome positioning is an active process that requires constant 'pressure' of agonist-GR-recruited chromatin-modifying factor(s) rather than GR-DNA binding itself.

Animals↗
PubMed2001

Glucocorticoid hormone-induced receptor localization to the chromatin fibers formed on injected DNA in Xenopus oocytes.

Oocytes from Xenopus laevis have provided a model system for studying the dynamic changes that occur in chromatin during gene activation. We have reconstituted glucocorticoid receptor (GR) induced transcription from the mouse mammary tumor virus (MMTV) promoter by intranuclear injection of an MMTV-driven reporter and cytoplasmic injection of synthetic mRNA(GR) into Xenopus oocytes. Here we investigate the intranuclear distribution of injected DNA, which is assembled into chromatin. We show that this chromatin is organized as an intranuclear fibrous network. Unliganded GR is located in the cytosol and hormone triggers its nuclear translocation and association with the chromatin fibers. Furthermore, we analyze the intranuclear distribution of other factors involved in transcription from the MMTV promoter. Indirect immunofluorescence microscopy on cryostat-sectioned oocytes revealed that BRG1, which is a subunit of the SWI/SNF chromatin remodeling complex, as well as RNA polymerase II and recombinantly expressed Xenopus nuclear factor 1-B, are all associated with the endogenous chromosomes and the chromatin fibers formed on injected DNA. This association does not depend on specific DNA binding sites and appears to be nonspecific.

Active Transport, Cell Nucleus↗
PubMed2000

Hormone activation induces nucleosome positioning in vivo.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone.

Animals↗
PubMed1999

Increased nuclear factor 1 binding to its nucleosomal site mediated by sequence-dependent DNA structure.

The organization of DNA into chromatin is important in the regulation of transcription, by influencing the access of transcription factors to their DNA binding sites. Nuclear factor 1 (NF-1) is a transcription factor which binds to DNA constitutively and which interacts with its cognate DNA site with high affinity. However, this affinity is drastically reduced, approximately 100- to 300-fold, when the binding site is organized into a nucleosome. Here we demonstrate that the introduction of stretches of adenines of length 5 nt (A-tracts) on both sides of the NF-1 binding site has a distinct effect on NF-1 binding to a nucleosomal, but not to a free, NF-1 binding site. The position of the A-tracts, relative to the rotational phase of a synthetic DNA bending sequence, the TG-motif, decides whether the NF-1 affinity increases or decreases. The NF-1 binding affinity is seven times stronger when the flanking A-tracts are positioned out-of-phase with the TG-motif than it is when the A-tracts are positioned in-phase with the TG-motif. We demonstrate that this effect correlates with differences in DNA curvature and apparent histone octamer affinity. We conclude that DNA curvature influences the local histone-DNA contacts and hence the accessibility of the NF-1 site in a nucleosome context.

Base Sequence↗
PubMed1999

Characterization of a chromatin remodelling activity in Xenopus oocytes.

The yeast SWI2/SNF2 protein is a component of a large protein complex which is involved in the remodelling of chromatin during transcriptional activation. Several homologous complexes have been found in Drosophila and mammals. We have examined the expression of the SWI2/SNF2 homologue BRG1 in Xenopus laevis using two antisera originally raised against the C-terminus of the rat and the human BRG1 protein. These two antisera cross-reacted with a protein found in both Xenopus liver and Xenopus oocytes. The Xenopus BRG1-like protein is expressed throughout oogenesis (stages I-VI) and embryogenesis. By injecting an expression vector containing the full-length human BRG1 cDNA into Xenopus oocytes, the relative molecular weight (Mr) of the Xenopus BRG1-like protein was shown to be slightly lower than that of the human BRG1, 190 000 and 200 000, respectively. The Xenopus BRG1-like protein elutes at a Mr of approximately 2 000 000 on Superose HR6trade mark size-exclusion chromatography, indicating that it is part of a larger complex, as are all other known SWI/SNF proteins. Nucleosome remodelling activity was co-eluted with the BRG1 immunogenic activity in both ion-exchange and size-exclusion chromatography.

Animals↗
PubMed1997

Assays for transcription factors access to nucleosomal DNA.

Several promoters have been shown to have sequence specifically positioned nucleosomes that determine the architecture of the promoter. DNA binding proteins that regulate gene expression are in many cases known to bind to their cognate DNA segments organized within such positioned nucleosomes. It has become increasingly evident that the cooperation of chromatin and transcription factors results in an efficient and fine-tuned regulation of transcription. The first step in a gene induction event must be the access of transcription factors for the regulatory promoter/enhancer target sites. In this perspective it becomes interesting to evaluate the affinity of DNA binding proteins for their cognate binding site in a nucleosome context. Here we describe the preparation of nucleosome probe, a method for in vitro nucleosome reconstitution by salt dilution, purification of the reconstituted mononucleosomes, and characterization of the translational and rotational positions of the nucleosomal DNA. In addition, methods for affinity determination and characterization of protein-nucleosomal DNA interaction, such as methylation protection and methylation interference by dimethyl sulfate, quantitative DNase I footprinting, and electrophoretic mobility shift assay, are described.

Binding, Competitive↗
PubMed1997

The role of chromatin in transcriptional regulation.

Transcriptional activation is mediated by the facilitated binding of the basal transcription complex to the transcription start site of a promoter. The activation procedure involves protein-protein interactions between specific transcription factors and members of the basal transcription complex. However, since eukaryotic DNA is packaged with histones into nucleosomes the accessibility of the transcription factors is limited. In order to activate transcription, some of the specific transcription factors must have the capacity to bind to their binding sites when organized into nucleosomes. As a next step, the chromatin structure of the promoter needs to be decondensed in order to facilitate the binding of the basal transcription machinery. Recent data have addressed these issues and both binding of transcription factors to their chromatin binding site as well as transcription factor-induced chromatin remodelling have been demonstrated. In addition, factors that are candidates to mediate the chromatin remodelling have recently been identified and characterized. The ability of a transcription factor to recognize its cognate element in a nucleosome is an inheret property that differs among different transcription factors. The implications of the rotational and translational positioning of the DNA within a nucleosome on the accessibility of a transcription factor is described in this review. In addition, nucleosome rearrangement and juxtaposing in the context of transcriptional activation is also discussed.

Animals↗
PubMed1997

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

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.

Adenosine Triphosphate↗
PubMed1996

The affinity of nuclear factor 1 for its DNA site is drastically reduced by nucleosome organization irrespective of its rotational or translational position.

A DNA-bending sequence has been used for in vitro reconstitution of nucleosomes in order to direct a nuclear factor 1 (NF-1) binding site into different nucleosome positions. By this strategy nucleosomes were obtained that had one of two rotational positions of the NF-1 binding site, one oriented toward the periphery and the other toward the histone octamer, translationally positioned 50 and 45 base pairs, respectively, from the nucleosome dyad. The affinity of partially purified NF-1 for these nucleosomal targets was compared with its affinity for free DNA by dimethylsulfate methylation protection and DNase I footprinting assays. The binding affinity of NF-1 to all nucleosomal targets was reduced 100-300-fold compared with its affinity for free DNA. The two rotational settings of the NF-1 site showed the same binding affinity for NF-1 as did other nucleosome constructs in which the NF-1 binding site was translationally positioned from 10 to 40 base pairs from the nucleosome dyad. We conclude that the nucleosomal inhibition of NF-1 binding is an inherent characteristic of NF-1 since another transcription factor, the glucocorticoid receptor, is able to bind to its DNA site in a nucleosome.

Base Sequence↗
PubMed1995

Triple helix DNA alters nucleosomal histone-DNA interactions and acts as a nucleosome barrier.

Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specific and required the oligonucleotide to be present during in vitro nucleosome reconstitution. Binding of the triple helix oligonucleotide on a 199 bp mouse mammary tumour virus promoter DNA fragment with a centrally located triplex DNA resulted in interruption of histone-DNA contacts flanking the triplex DNA segment. When nucleosome reconstitution is carried out on a longer, 279 bp DNA fragment with an asymmetrically located triplex site, nucleosome formation occurred at the border of the triple helical DNA. In this case the triplex DNA functioned as a nucleosome barrier. We conclude that triplex DNA cannot be accommodated within a nucleosome context and thus may be used to site-specifically manipulate nucleosome organization.

Base Sequence↗
PubMed1995

Accessibility of a glucocorticoid response element in a nucleosome depends on its rotational positioning.

Gene expression requires binding of transcription factors to their cognate DNA response elements, the latter being often integrated into sequence-specifically positioned nucleosomes. To investigate the constraints imposed on factor-DNA recognition by the nucleosomal organization, we studied the binding of glucocorticoid receptor to a single glucocorticoid response element (GRE) displaying four different rotational frames in three different translational positions in reconstituted nucleosomes. We demonstrate that rotational setting of the GRE per se is important for its accessibility. Furthermore, the effects of rotational positioning of the GRE are different for different translational positions of the GRE in the nucleosome. A GRE placed near the nucleosomal dyad is totally blocked by rotating it 180 degrees so that the major groove of the GRE faces the histone octamer. If, on the other hand, the GRE is placed about 40 bp from the nucleosome dyad, then the 180 degrees rotation of the GRE still allows glucocorticoid receptor binding, albeit with a sixfold lower affinity than the peripherally oriented GRE. This suggests that both the rotational positioning and the translational positioning function as a framework for transcription factor response elements in gene regulation.

Base Sequence↗
PubMed1993

The glucocorticoid receptor acts as an antirepressor in receptor-dependent in vitro transcription.

Glucocorticoid-receptor-dependent and glucocorticoid-response-element-dependent in vitro transcription was established using a crude nuclear extract and purified glucocorticoid receptor from rat liver. The capacity of glucocorticoid receptor to stimulate in vitro transcription was only detectable when basal transcription, i.e. transcription in the absence of glucocorticoid receptor, had been repressed. Transcriptional repression was achieved either by adding purified histone H1, or by lowering the amount of DNA template relative to the amount of crude nuclear extract. Glucocorticoid receptor caused a 1.1 +/- 0.7-fold stimulation of transcription from the mouse-mammary-tumor-virus promoter when basal transcription was not repressed, and a 7.0 +/- 1.5-fold stimulation when basal transcription had been repressed by addition of histone H1. Similar results were obtained when using a minimal promoter consisting of two glucocorticoid-response elements and a TATA box. Our data suggest that glucocorticoid receptor stimulates in vitro transcription by an antirepression mechanism.

Animals↗
PubMed1993

Translational positioning of a nucleosomal glucocorticoid response element modulates glucocorticoid receptor affinity.

Positioned nucleosomes are often found in enhancer/promoter regions where they confer defined positioning of trans-active-factor response element(s) relative to the histone octamer. Here, we address how this affects factor/response element recognition. We used 165-bp DNA segments containing one glucocorticoid response element (GRE) and rat liver core histones to reconstitute nucleosomes in vitro. The GREs in these nucleosomes were held in identical helical settings but different translational positions. This was achieved by placing the GRE within or at the flank of a 95-bp DNA-bending sequence. Glucocorticoid receptor (GR)-binding experiments demonstrated that a GRE in free DNA has a 2.5-fold higher affinity for GR than the nucleosomal GRE when positioned 40 bp from the nucleosome dyad. A nucleosomal GRE positioned 40 bp from the nucleosome dyad, on the other hand, binds GR 4.3-fold better as compared to an identical GRE positioned 20 bp from the dyad and 1.4-fold better than a GRE positioned at the dyad. Interruption of the DNA-bending sequence, by a 5-bp AT segment next to a nucleosomal GRE positioned 20 bp from the dyad, restores GR affinity to the same level as when the GRE is placed 40 bp from the dyad. The effect on GR/GRE affinity either by different positioning within the 95-bp bending sequence or by introducing the 5-bp AT-segment is seen only in a nucleosomal context. We conclude that a translationally positioned nucleosome can modulate the affinity of a trans-active factor for its target response element.

Animals↗
PubMed1991

The glucocorticoid receptor in homodimeric and monomeric form visualised by electron microscopy.

The purified glucocorticoid receptor (GR) from rat liver has been visualised by electron microscopy. The specimens were prepared by spreading on thin carbon support and negatively stained using uranyl acetate. Two forms of GR, the monomeric and the dimeric forms, were identified based on size, chromatographic distribution, and DNA binding properties. The GR monomer consists of two globular domains of slightly different size with a thinner connecting domain in between. In the absence of DNA the dimeric GR has a characteristic four-leaf clover structure. The size and appearance of this structure is consistent with two GR subunits arranged in a side-by-side fashion. Monomeric and dimeric GR specifically bound to DNA are also shown.

Animals↗
PubMed1991

Inhibition of chromatin assembly in Xenopus oocytes correlates with derepression of the mouse mammary tumor virus promoter.

The mouse mammary tumor virus (MMTV) promoter is positively regulated by glucocorticoid hormone via binding of glucocorticoid receptor to a specific response element. Upon addition of hormone, a nucleosome containing the glucocorticoid response element is removed or structurally altered, suggesting that the nucleosome interferes with transcription. Accordingly, inhibition of chromatin assembly should relieve the repression and result in an increased constitutive activity. We have tested this hypothesis by injecting nonspecific competitor DNA into Xenopus laevis oocytes to titrate endogenous histones. The coinjection of competitor DNA altered chromatin structure: nucleosomal ladders produced by micrococcal nuclease were disrupted, and the unique helical setting of the MMTV promoter in a nucleosome was lost, as shown by in situ DNase I footprinting. Basal MMTV transcription was drastically increased by competitor DNA, whereas a coinjected, constitutively active adenovirus 2 major late promoter was not stimulated. These results show that the uninduced MMTV promoter is under negative control and provide direct support for the theory that the nucleosomal organization maintains the repression of this promoter in its uninduced state.

Adenoviridae↗
PubMed1990

Quantitative analysis of the glucocorticoid receptor-DNA interaction at the mouse mammary tumor virus glucocorticoid response element.

Purified glucocorticoid receptor (GR) from rat liver was used for a quantitative analysis of the protein-DNA interaction at specific GR-binding segments within the 5'-long terminal repeat of the mouse mammary tumor virus. A truncated receptor was generated and used to demonstrate formation of heterodimeric GR, which furthermore was shown to be in rapid equilibrium with receptor-monomer. The relative affinity for GR binding to specific GR sites versus random calf thymus DNA was approximately 2 x 10(3). At equilibrium a free GR concentration of 3 x 10(-10) M was required for half-maximal saturation of the two functionally important DNA sites within the mouse mammary tumor virus 5'-long terminal repeat. Although these two DNA segments act synergistically in mediating hormonal response, we did not detect cooperative GR binding to these regions in vitro. However, GR bound cooperatively within the downstream binding region. Similarly, GR was unable to facilitate factor binding to a neighboring nuclear factor 1 site, another essential element in the promoter. In contrast, nuclear factor 1 binding was inhibited slightly by GR.

Animals↗
PubMed1990

Protein-protein contacts in the glucocorticoid receptor homodimer influence its DNA binding properties.

We have investigated the influence of the N-terminal domain of the 94-kDa glucocorticoid receptor on the DNA:receptor interaction. An alpha-chymotrypsin-induced 39-kDa receptor fragment, containing the hormone and DNA binding domains, binds DNA with a reduced specificity compared to the intact 94-kDa receptor. Various footprinting assays did not reveal any qualitative differences when comparing the DNA contact points made by the two different receptor entities. Like the intact receptor, the 39-kDa receptor fragment binds as a dimer to DNA. Glutaraldehyde cross-linking demonstrated a difference in the protein:protein contacts of the two homodimers. Furthermore, the dimeric 94-kDa receptor did not recognize a half-DNA site, while the dissociated 94-kDa receptor dimer and the dimeric 39-kDa receptor fragment allowed binding to such a site. These results suggest that the loss of the N-terminal domain of the receptor affects the steric arrangement and/or rigidity of the two DNA binding domains of the receptor homodimer, resulting in a decreased DNA binding specificity of the 39-kDa receptor fragment.

Animals↗