scholarly journals Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition

2000 ◽  
Vol 20 (17) ◽  
pp. 6466-6475 ◽  
Author(s):  
Terace M. Fletcher ◽  
Byung-Woo Ryu ◽  
Christopher T. Baumann ◽  
Barbour S. Warren ◽  
Gilberto Fragoso ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Dynamic Properties ◽  
Nuclear Extract ◽  
Hela Nuclear Extract ◽  
Mouse Mammary Tumor ◽  
Multistep Process ◽  
Chromatin Structural ◽  
Tumor Virus ◽  
Mmtv Ltr

ABSTRACT Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633–3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a “hit-and-run” mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262–1264, 2000).

scholarly journals The HSA Domain of BRG1 Mediates Critical Interactions Required for Glucocorticoid Receptor-Dependent Transcriptional Activation In Vivo

2007 ◽  
Vol 28 (4) ◽  
pp. 1413-1426 ◽  
Author(s):  
Kevin W. Trotter ◽  
Hua-Ying Fan ◽  
Melissa L. Ivey ◽  
Robert E. Kingston ◽  
Trevor K. Archer
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Critical Factor ◽  
Receptor Complexes ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Eukaryotic Dna

ABSTRACT The packaging of eukaryotic DNA into chromatin can create an impediment to transcription by hindering binding of essential factors required for transcription. The mammalian SWI/SNF remodeling complex has been shown to alter local chromatin structure and facilitate recruitment of transcription factors. BRG1 (or hBrm), the central ATPase of the human SWI/SNF complex, is a critical factor for the functional activity of nuclear receptor complexes. Analysis using BRG1/SNF2h chimeras suggests BRG1 may contain previously uncharacterized functional motifs important for SWI/SNF. To identify these regions, BRG1 truncation and deletion mutants were designed, characterized, and utilized in a series of assays to evaluate transcriptional activation and chromatin remodeling by the glucocorticoid receptor. We identified a domain within the N terminus of BRG1 that mediates critical protein interactions within SWI/SNF. We find the HSA domain of BRG1 is required to mediate the interaction with BAF250a/ARID1A and show this association is necessary for transcriptional activation from chromatin mouse mammary tumor virus or endogenous promoters in vivo. These studies suggest BAF250a is a necessary facilitator of BRG1-mediated chromatin remodeling required for SWI/SNF-dependent transcriptional activation.

scholarly journals ATP-Dependent Mobilization of the Glucocorticoid Receptor during Chromatin Remodeling

2002 ◽  
Vol 22 (10) ◽  
pp. 3255-3263 ◽  
Author(s):  
Terace M. Fletcher ◽  
Nianqing Xiao ◽  
Gisele Mautino ◽  
Christopher T. Baumann ◽  
Ronald Wolford ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Direct Interaction ◽  
Nuclear Extract ◽  
Magnetic Bead ◽  
Tumor Virus ◽  
Nuclear Factor 1

ABSTRACT Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATP-dependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.

scholarly journals CDP Binding to Multiple Sites in the Mouse Mammary Tumor Virus Long Terminal Repeat Suppresses Basal and Glucocorticoid-Induced Transcription

2002 ◽  
Vol 76 (5) ◽  
pp. 2168-2179 ◽  
Author(s):  
Quan Zhu ◽  
Jaquelin P. Dudley
Keyword(s):  
Mammary Gland ◽  
Reporter Gene ◽  
Binding Sites ◽  
Mouse Mammary Tumor Virus ◽  
Mammary Gland Development ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Ltr ◽  
Gland Development

ABSTRACT Mouse mammary tumor virus (MMTV) is transcribed at high levels in the lactating mammary gland to ensure transmission of virus from the milk of infected female mice to susceptible offspring. We previously have shown that the transcription factor CCAAT displacement protein (CDP) is expressed in high amounts in virgin mammary gland, yet DNA-binding activity for the MMTV long terminal repeat (LTR) disappears as mammary tissue differentiates during lactation. CDP is a repressor of MMTV expression and, therefore, MMTV expression is suppressed during early mammary gland development. In this study, we have shown using DNase I footprinting and electrophoretic mobility shift assays that there are at least five CDP-binding sites in the MMTV LTR upstream of those previously described in the promoter-proximal negative regulatory element (NRE). Single mutations in two of these upstream sites (+691 or +692 and +735 relative to the first base of the LTR) reduced CDP binding to the cognate sites and elevated reporter gene expression from the full-length MMTV LTR. Combination of a mutation in the promoter-distal NRE with a mutation in the proximal NRE gave approximately additive increases in LTR-reporter gene activity, suggesting that these binding sites act independently. Mutations in several different CDP-binding sites allowed elevation of reporter gene activity from the MMTV promoter in the absence and presence of glucocorticoids, hormones that contribute to high levels of MMTV transcription during lactation by activation of hormone receptor binding to the LTR. In addition, overexpression of CDP in transient-transfection assays suppressed both basal and glucocorticoid-induced LTR-mediated transcription in a dose-dependent manner. These data suggest that multiple CDP-binding sites contribute independently to regulate binding of positive factors, including glucocorticoid receptor, to the MMTV LTR during mammary gland development.

scholarly journals Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice.

1990 ◽  
Vol 10 (11) ◽  
pp. 5822-5829 ◽  
Author(s):  
S R Ross ◽  
C L Hsu ◽  
Y Choi ◽  
E Mok ◽  
J P Dudley
Keyword(s):  
Transgenic Mice ◽  
Mouse Mammary Tumor Virus ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Ltr ◽  
Inappropriate Expression

Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene.

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