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A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor.

ABSTRACT: Several of the SNF and SWI genes of Saccharomyces cerevisiae code for proteins believed to assist transcriptional activators by relieving nucleosome repression. One of these proteins, SNF2/SWI2, has a homologue in Drosophila, a regulator of homeotic genes known as brahma or brm. In this report, we show that a counterpart of SNF2/SWI2 also exists in mice and humans. The human protein, designated hbrm, is a 180 kDa nuclear factor that can function as a transcriptional activator when fused to a heterologous DNA binding domain. The mouse homologue of hbrm is expressed in all mouse organs tested while hbrm was detected in some but not all investigated human cell lines. In cells failing to express the endogenous gene, transfected hbrm cooperates with the glucocorticoid receptor (GR) in transcriptional activation. However, hbrm had no effect on the activity of several other transcription factors, including the homeoprotein HNF-1. The co-operation between hbrm and GR required the DNA binding domain of GR and two separated regions of the hbrm protein, including a domain with homology to known helicases.

SUBMITTER: Muchardt C

PROVIDER: S-EPMC413724 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor.

Muchardt C C Yaniv M M

The EMBO journal 19931101 11

Several of the SNF and SWI genes of Saccharomyces cerevisiae code for proteins believed to assist transcriptional activators by relieving nucleosome repression. One of these proteins, SNF2/SWI2, has a homologue in Drosophila, a regulator of homeotic genes known as brahma or brm. In this report, we show that a counterpart of SNF2/SWI2 also exists in mice and humans. The human protein, designated hbrm, is a 180 kDa nuclear factor that can function as a transcriptional activator when fused to a het ...[more]

PMID: 8223438

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Project description:Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryote Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so is unclear. We used multi-wavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the E. coli transcription cycle. In our experiments, RapA at < 5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post-termination complex (PTC) -- consisting of core RNA polymerase (RNAP) bound to dsDNA -- and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription re-initiation in proteobacterial genomes.SignificanceRNA synthesis is an essential conduit of genetic information in all organisms. After transcribing an RNA, the bacterial RNA polymerase (RNAP) must be reused to make subsequent RNAs, but the steps that enable RNAP reuse are unclear. We directly observed the dynamics of individual molecules of fluorescently labeled RNAP and the enzyme RapA as they colocalized with DNA during and after RNA synthesis. Our studies show that RapA uses ATP hydrolysis to remove RNAP from DNA after the RNA is released from RNAP and reveal essential features of the mechanism by which this removal occurs. These studies fill in key missing pieces in our current understanding of the events that occur after RNA is released and that enable RNAP reuse.

Project description:BackgroundPreviously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes.MethodologyUsing fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase ADomain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized.SignificanceThe use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.

Dataset Information

A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor.

Publications

A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor.

Similar Datasets

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