scholarly journals Reconstitution of Glucocorticoid Receptor-Dependent Transcription In Vivo

2004 ◽  
Vol 24 (8) ◽  
pp. 3347-3358 ◽  
Author(s):  
Kevin W. Trotter ◽  
Trevor K. Archer
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Factor Loading ◽  
Baf Complex ◽  
Functional Remodeling ◽  
Chromatin Template ◽  
Necessary And Sufficient

ABSTRACT We developed a model system to study glucocorticoid receptor (GR)-mediated chromatin remodeling by the BRG1 complex. Introduction of the BRG1 ATPase into the SW-13 cell line initiates the formation of a functional remodeling complex. This complex is able to induce transcriptional activation from a transiently transfected promoter with wild-type and chromatin-remodeling-deficient BRG1 mutants, suggesting that the complex possesses a coactivator function independent from remodeling. Transactivation from a chromatin template requires the BRG1 remodeling function, which induces regions of hypersensitivity and transcription factor loading onto the integrated MMTV promoter. We report that BRG1 remodeling activity is required for GR-mediated transactivation and that this activity cannot be replaced by other ATP-dependent remodeling proteins. Further characterization of the BRG1-associated factors (BAFs) present in these cells (for example, the expression of BAF250 but not BAF180) reveals that the BAF complex rather than the polybromo-associated BAF complex is the necessary and sufficient chromatin-remodeling component with which the receptor functions in vivo. These results in conjunction with previous findings demonstrate that the GR functions with multiple forms of the SWI/SNF complex in vivo.

scholarly journals A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex

2000 ◽  
Vol 20 (23) ◽  
pp. 8879-8888 ◽  
Author(s):  
Zuqin Nie ◽  
Yutong Xue ◽  
Dafeng Yang ◽  
Sharleen Zhou ◽  
Bonnie J. Deroo ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Remodeling ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Dna Interaction ◽  
Protein Protein Interactions ◽  
Baf Complex ◽  
Chromatin Remodeling Complexes

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc fromSaccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

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 An In Vitro System Recapitulates Chromatin Remodeling at the PHO5 Promoter

1999 ◽  
Vol 19 (4) ◽  
pp. 2817-2827 ◽  
Author(s):  
Elizabeth S. Haswell ◽  
Erin K. O’Shea
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Structure ◽  
Nuclear Extract ◽  
In Vitro System ◽  
Chromatin Remodeling Complex ◽  
Promoter Dna ◽  
Pho5 Promoter

ABSTRACT The Saccharomyces cerevisiae gene PHO5 is an excellent system with which to study regulated changes in chromatin structure. The PHO5 promoter is packaged into four positioned nucleosomes under repressing conditions; upon induction, the structure of these nucleosomes is altered such that the promoter DNA becomes accessible to nucleases. We report here the development and characterization of an in vitro system in which partially purified PHO5 minichromosomes undergo promoter chromatin remodeling. Several hallmarks of thePHO5 chromatin transition in vivo were reproduced in this system. Chromatin remodeling of PHO5minichromosomes required the transcription factors Pho4 and Pho2, was localized to the promoter region of PHO5, and was independent of the chromatin-remodeling complex Swi-Snf. In vitro chromatin remodeling also required the addition of fractionated nuclear extract and hydrolyzable ATP. This in vitro system should serve as a useful tool for identifying the components required for this reaction and for elucidating the mechanism by which the PHO5promoter chromatin structure is changed.

scholarly journals Identification and characterization of Drosophila relatives of the yeast transcriptional activator SNF2/SWI2.

1994 ◽  
Vol 14 (4) ◽  
pp. 2225-2234 ◽  
Author(s):  
L K Elfring ◽  
R Deuring ◽  
C M McCallum ◽  
C L Peterson ◽  
J W Tamkun
Keyword(s):  
Transcriptional Activation ◽  
Slow Growth ◽  
Transcriptional Activator ◽  
Yeast Cells ◽  
Homeotic Genes ◽  
Dependent Atpase ◽  
Functional Homolog ◽  
Identification And Characterization

The Drosophila brahma (brm) gene encodes an activator of homeotic genes that is highly related to the yeast transcriptional activator SWI2 (SNF2), a potential helicase. To determine whether brm is a functional homolog of SWI2 or merely a member of a family of SWI2-related genes, we searched for additional Drosophila genes related to SWI2 and examined their function in yeast cells. In addition to brm, we identified one other Drosophila relative of SWI2: the closely related ISWI gene. The 1,027-residue ISWI protein contains the DNA-dependent ATPase domain characteristic of the SWI2 protein family but lacks the three other domains common to brm and SWI2. In contrast, the ISWI protein is highly related (70% identical) to the human hSNF2L protein over its entire length, suggesting that they may be functional homologs. The DNA-dependent ATPase domains of brm and SWI2, but not ISWI, are functionally interchangeable; a chimeric SWI2-brm protein partially rescued the slow growth of swi2- cells and supported transcriptional activation mediated by the glucocorticoid receptor in vivo in yeast cells. These findings indicate that brm is the closest Drosophila relative of SWI2 and suggest that brm and SWI2 play similar roles in transcriptional activation.

scholarly journals Maximal Induction of a Subset of Interferon Target Genes Requires the Chromatin-Remodeling Activity of the BAF Complex

2002 ◽  
Vol 22 (18) ◽  
pp. 6471-6479 ◽  
Author(s):  
Hong Liu ◽  
Hyeog Kang ◽  
Rui Liu ◽  
Xin Chen ◽  
Keji Zhao
Keyword(s):  
Chromatin Remodeling ◽  
Target Genes ◽  
Transcription Activator ◽  
Baf Complex ◽  
Antiproliferative Effects ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Maximal Induction ◽  
Antiproliferative Activities

ABSTRACT The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-α). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-α induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.

scholarly journals RBP-L, a transcription factor related to RBP-Jkappa.

1997 ◽  
Vol 17 (5) ◽  
pp. 2679-2687 ◽  
Author(s):  
S Minoguchi ◽  
Y Taniguchi ◽  
H Kato ◽  
T Okazaki ◽  
L J Strobl ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Notch Receptor ◽  
In Vitro Assays ◽  
Functional Interaction ◽  
Protein Protein Interaction ◽  
Isolation And Characterization ◽  
Signalling Molecule

RBP-Jkappa is a sequence-specific DNA binding protein which plays a central role in signalling downstream of the Notch receptor by physically interacting with its intracellular region. Although at least four Notch genes exist in mammals, it is unknown whether each Notch requires a specific downstream signalling molecule. Here we report isolation and characterization of a mouse RBP-Jkappa-related gene named RBP-L that is expressed almost exclusively in lung, in contrast to the ubiquitous expression of RBP-Jkappa. For simplicity, we propose to call RBP-Jkappa RBP-J. The RBP-L protein bound to a DNA sequence almost identical to that of RBP-J. Surprisingly, RBP-L did not interact with any of the known four mouse Notch proteins. Although we found that RBP-L and EBNA-2 cooperated in transcriptional activation, they did not show significantly strong protein-protein interaction that can be detected by several in vivo and in vitro assays. This is again in contrast to physical association of RBP-J with EBNA-2. Several models to explain functional interaction between RBP-L and EBNA-2 are discussed.

scholarly journals Interaction of Papillomavirus E2 Protein with the Brm Chromatin Remodeling Complex Leads to Enhanced Transcriptional Activation

2006 ◽  
Vol 81 (5) ◽  
pp. 2213-2220 ◽  
Author(s):  
R. Ajay Kumar ◽  
Samisubbu R. Naidu ◽  
Xiaoyu Wang ◽  
Anthony N. Imbalzano ◽  
Elliot J. Androphy
Keyword(s):  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Potential Interaction ◽  
Transactivation Domain ◽  
Reporter System ◽  
Barr Virus ◽  
Amino Terminal ◽  
Chromatin Remodeling Complex ◽  
Epstein Barr

ABSTRACT Papillomavirus E2 is a sequence-specific DNA binding protein that regulates transcription and replication of the viral genome. The transcriptional activities of E2 are typically evaluated by transient transfection of nonreplicating E2-dependent reporters. We sought to address whether E2 activates transcription in an episomal context and its potential interaction with the chromatin remodeling proteins. Using an Epstein-Barr virus-based episomal reporter, we demonstrate that E2 stimulates transcription from an E2-dependent promoter in a chromatin context. This activation is enhanced by the presence of proteins associated with SWI/SNF complexes, which are ATP-dependent chromatin remodeling enzymes. We show that exogenous expression of the Brm ATPase enhances E2 activity in SWI/SNF-deficient cell lines and that the amino-terminal transactivation domain of E2 mediates association with the Brm complex in vivo. Using chromatin immunoprecipitation assays, we demonstrate that Brm enhances promoter occupancy by E2 in an episomal context. Our results demonstrate that E2 activates transcription from an episomal reporter system and reveal a novel property of E2 in collaborating with the Brm chromatin remodeling complex in enhancing transcriptional activation.

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