The SWI/SNF chromatin remodeling complexes regulate the transcription of many genes by remodeling nucleosomes at promoter regions. In Drosophila, SWI/SNF plays an important role in ecdysone-dependent transcription regulation. Studies in human cells suggest that Brahma (Brm), the ATPase subunit of SWI/SNF, regulates alternative pre-mRNA splicing by modulating transcription elongation rates. We describe, here, experiments that study the association of Brm with transcribed genes in Chironomus tentans and Drosophila melanogaster, the purpose of which was to further elucidate the mechanisms by which Brm regulates pre-mRNA processing. We show that Brm becomes incorporated into nascent Balbiani ring pre-mRNPs co-transcriptionally and that the human Brm and Brg1 proteins are associated with RNPs. We have analyzed the expression profiles of D. melanogaster S2 cells in which the levels of individual SWI/SNF subunits have been reduced by RNA interference, and we show that depletion of SWI/SNF core subunits changes the relative abundance of alternative transcripts from a subset of genes. This observation, and the fact that a fraction of Brm is not associated with chromatin but with nascent pre-mRNPs, suggest that SWI/SNF affects pre-mRNA processing by acting at the RNA level. Ontology enrichment tests indicate that the genes that are regulated post-transcriptionally by SWI/SNF are mostly enzymes and transcription factors that regulate postembryonic developmental processes. In summary, the data suggest that SWI/SNF becomes incorporated into nascent pre-mRNPs and acts post-transcriptionally to regulate not only the amount of mRNA synthesized from a given promoter but also the type of alternative transcript produced.
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.DNA in eukaryotic cells associates with proteins called histones to form nucleosomes, which together with nonhistone proteins form a higher-order structure, chromatin (22). It is now well established that chromatin not only provides a DNA storage function but is also involved in gene regulation (41,63). When the synthesis of histone H4 is inhibited in yeast cells, several genes which are tightly regulated are expressed in a constitutive manner (14). The mouse mammary tumor virus (MMTV) promoter, which is repressed in the absence of glucocorticoid hormone, becomes constitutively expressed when the nucleosome density is decreased by coinjection of competitor DNA, as shown in Xenopus oocytes (43). Injection of single-stranded DNA into Xenopus oocytes leads to nucleosome assembly coupled to DNA synthesis. This results in a tighter chromatin structure which confers a more stringent repression of transcription than chromatin formed on DNA injected in the double-stranded form (2). Several studies suggest that chromatin acts both by excluding certain upstreamgene-specific transcription factors from their recognition sites (1, 4) and by inhibiting access of the basic transcription machinery to the transcriptional initiation site (17,30,33,64).Regulatory regions of many inducible genes in mammals and yeast have positioned nucleosomes. Examples include the MMTV promoter (53), the...
A simple method is described for the separation and quantification of the subunits of GSH transferases present in rat tissue extracts. This method, involving GSH-agarose affinity chromatography followed by reverse-phase h.p.l.c., is rapid and sufficiently sensitive to measure 5 micrograms of each subunit in a mixture. Examples are given of its application to extracts of rat kidney, adrenal, testicular interstitial cells and seminiferous tubules. The analysis of seminiferous tubules indicates that the technique may be of value for the identification of novel subunits. Preliminary separations of subunits from human GSH transferases are also described.
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