Mammalian SWI-SNF Complexes Contribute to Activation of the hsp70 Gene

Abstract

ATP-dependent chromatin-remodeling complexes are conserved among all eukaryotes and function by altering nucleosome structure to allow cellular regulatory factors access to the DNA. Mammalian SWI-SNF complexes contain either of two highly conserved ATPase subunits: BRG1 or BRM. To identify cellular genes that require mammalian SWI-SNF complexes for the activation of gene expression, we have generated cell lines that inducibly express mutant forms of the BRG1 or BRM ATPases that are unable to bind and hydrolyze ATP. The mutant subunits physically associate with at least two endogenous members of mammalian SWI-SNF complexes, suggesting that nonfunctional, dominant negative complexes may be formed. We determined that expression of the mutant BRG1 or BRM proteins impaired the ability of cells to activate the endogenous stress response gene hsp70 in response to arsenite, a metabolic inhibitor, or cadmium, a heavy metal. Activation of hsp70 by heat stress, however, was unaffected. Activation of the heme oxygenase 1 promoter by arsenite or cadmium and activation of the cadmium-inducible metallothionein promoter also were unaffected by the expression of mutant SWI-SNF components. Analysis of a subset of constitutively expressed genes revealed no or minimal effects on transcript levels. We propose that the requirement for mammalian SWI-SNF complexes in gene activation events will be specific to individual genes and signaling pathways.The packaging of eukaryotic DNA into nucleosomes and higher order chromatin structure presents cells with a significant barrier to DNA utilization and necessitates mechanisms by which chromatin structure can be modified so that transcription can occur. Many multiprotein complexes with the ability to modify chromatin structure have been identified. These include histone acetyltransferases and deacetylases, which directly modify histone tail domains, and a class of energy-dependent enzymes that utilize ATP hydrolysis to alter nucleosome structure (reviewed in references 23, 30, 32, 34, 70, 83, and 84). The ATP-dependent chromatin remodeling complexes are conserved among eukaryotes, they share a related subunit that possesses DNA-stimulated ATPase activity, and each has been demonstrated to alter nucleosome structure in vitro in an ATP-dependent manner. Most of these complexes can be classified into two groups, those containing homologues of the yeast SWI2-SNF2 ATPase subunit, including yeast SWI-SNF (7, 12, 55), human SWI-SNF (hSWI-SNF) (24, 35, 82), yeast RSC (8), and Drosophila BRM complexes (54, 71), and those containing homologues of the Drosophila imitation-switch (ISWI) ATPase gene (16), including yeast ISW1 and ISW2 (76), human RSF (39), and the Drosophila NURF, CHRAC, and ACF complexes (25,75,78). A third group can be defined by Xenopus and human complexes containing the Mi2 protein, a related ATPase found in association with histone deacetylase activity (72,81,87,90).Although members of the ATP-dependent class of chromatin remodelers facilitate alterations in nucleosome structure in ...

Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

“…A strong but diffuse nucleoplasmic staining pattern was noted for ISWI, but unlike BRG1 or BRM, this remodeling protein was not enriched at the array upon dexamethasone treatment, suggesting that hormone-dependent activation of the MMTV promoter involves the selective and class-specific recruitment of remodeling proteins (Figure 2, T and X). The involvement of multiple members of the SWI/ SNF family in transcriptional regulation is similar to what has been previously described for the hsp70 promoter (de la Serna et al, 2000). BRG1 and BRM may have separate and distinct roles in the transcriptional process; this is yet to be determined.…”

“…ATPase-deficient forms of BRG1 or BRM (BRG1-K-R or BRM-K-R) contain a lysine-to-arginine mutation in the ATP binding pocket (Figure 1, A and B) that abrogates ATP hydrolysis but retains the ability to efficiently incorporate into multisubunit SWI/SNF-like complexes (Rayasam et al, 2005). Consequently, when overexpressed, these mutants function as dominant negatives (de la Serna et al, 2000). Transfection of SW13 cells with ATPase-deficient forms of BRG1 or BRM compromised transcription dramatically (to eightfold), suggesting that optimal MMTV transcription in SW13 cells required a chromatin remodeling competent BRG1 or BRM ( Figure 1C).…”

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1

“…NIH 3T3 cells expressing either antisense PRMT5 or FLAG-tagged catalytically inactive BRG1 (Fl-MutBRG1) were grown as described previously (15,24). To establish HeLa S3 cell lines that stably express epitope-tagged wild type or mutant PRMT7, 70 -80% confluent plates were transfected with 2 g of pBabe/Fl-PRMT7 or pBabe/Fl-Mut/PRMT7 plasmid for 5 h using Lipofectamine 2000 (Invitrogen).…”

Selective and antagonistic functions of SWI/SNF and Mi-2β nucleosome remodeling complexes during an inflammatory response

“…The dominant-negative BRG1 contains a single point mutation in the ATP-binding domain that permits proper folding and complex assembly, but disrupts nucleosome remodeling (de La Serna et al 2000). Flag-tagged wild-type and dominant-negative BRG1 (BRG1 WT and BRG1 DN) were stably expressed in J774 cells by retroviral transduction (Fig.…”

“…For this reason, in vivo studies of SWI/SNF functions in mammalian cells have relied more strongly on dominant-negative approaches and on the SW13 cell line, which has adapted to growth in the absence of SWI/SNF complexes, than on mice or cells with disrupted SWI/ SNF alleles (de La Serna et al 2000;Liu et al 2001;Chi et al 2003;Chi 2004). As an alternative strategy for performing loss-of-function studies, we disrupted expression of remodeling complexes by retroviral delivery of small interfering RNAs (siRNAs) and focused on gene activation in response to LPS, an acute extracellular stimulus.…”