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 cytochrome P450 1B1 gene (CYP1B1) is expressed constitutively and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the human breast adenocarcinoma cell line MCF-7 but not in the human hepatoma cell line HepG2. Genomic DNA isolated from both cell lines was digested with the methylation-sensitive restriction enzyme isoschizomers MspI and HpaII, and subjected to Southern analysis with a probe for the CYP1B1 promoter/enhancer region. Although differences were observed in methylation patterns for the CYP1B1 gene from MCF-7 and HepG2 cells, treatment with the demethylating agent 5-azacytidine (10 microM for 6 days) did not activate CYP1B1 mRNA expression in HepG2 cells. Furthermore, treatment with the histone deacetylase inhibitor trichostatin A (100 nM for 24 h) did not activate CYP1B1 mRNA expression in HepG2 cells. Comparative analysis of the constitutive expression of luciferase/1B1 reporter constructs containing a series of deletions in the 5' enhancer region indicated that in MCF-7 cells the region from -987 to -732 (relative to the transcription start site) was necessary for maximal levels of activity. Mutation of the aryl hydrocarbon receptor response elements (dioxin response elements) in this region showed that the dioxin response elements located at -833 is essential for constitutive gene expression in MCF-7 cells. In HepG2 cells, reporter gene activity was at least equal or greater than the activity observed in MCF-7 cells, which is in marked contrast to the expression of the native CYP1B1 gene. Taken together these findings indicate that the observed cell-specific differences in CYP1B1 constitutive expression are not mediated by DNA promoter/enhancer methylation, but are likely due to either 1) inaccessibility of the 5'-enhancer region in HepG2 cells to transcriptional activators due to a higher order chromatin structure that does not involve histone acetylation, or 2) the action of a repressor protein at cis-elements located outside of the -2296 to +25 region examined with the CYP1B1 reporter constructs. Furthermore, at least one of the dioxin response elements in the enhancer region is required for constitutive expression of CYP1B1.