No abstract

The functions of the SAGA and SWI/SNF complexes are interrelated and can form stable "epigenetic marks" on promoters in vivo. Here we show that stable promoter occupancy by SWI/SNF and SAGA in the absence of transcription activators requires the bromodomains of the Swi2/Snf2 and Gcn5 subunits, respectively, and nucleosome acetylation. This acetylation can be brought about by either the SAGA or NuA4 HAT complexes. The bromodomain in the Spt7 subunit of SAGA is dispensable for this activity but will anchor SAGA if it is swapped into Gcn5, indicating that specificity of bromodomain function is determined in part by the subunit it occupies. Thus, bromodomains within the catalytic subunits of SAGA and SWI/SNF anchor these complexes to acetylated promoter nucleosomes.

To investigate the function of SWI/SNF in site-specific chromatin remodeling at promoters, we have used a purified system to analyze its distribution, function, and retention following recruitment by a sequence-specific transcription activator. Activator recruitment of SWI/SNF bound the complex to promoter proximal nucleosomes and led to localized nucleosome disruption. However, retention of SWI/SNF on the promoter required either the continued binding of the transcription activator or acetylated histones. Histone acetylation by either the SAGA or NuA4 HAT complexes increased the retention of SWI/SNF on the promoter. These data illustrate a functional link between HAT complexes and the SWI/SNF chromatin remodeling complex and provide a mechanistic basis for the ordered recruitment of these complexes.

The yeast SWI/SNF complex is required for the transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP-dependent reaction. In this study, we show that the complex stimulated in vitro transcription from nucleosome templates in an activation domain-dependent manner. Transcription stimulation by SWI/SNF required an activation domain with which it directly interacts. The acidic activation domains of VP16, Gcn4, Swi5, and Hap4 interacted directly with the purified SWI/SNF complex and with the SWI/SNF complex in whole-cell extracts. The similarity of activation domain interactions and transcriptional stimulation between SWI/SNF and the SAGA histone acetyltransferase complex may account for their apparent overlapping functions in vivo.

We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photocross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains.The yeast SWI/SNF complex alters nucleosome structure in an ATP-dependent reaction, and SWI/SNF activity can lead to the relief of chromatin-mediated repression of transcription (for reviews, see references 30, 50, 64, and 70). SWI/SNF is part of a large family of ATP-dependent chromatin remodeling enzymes, which have been purified and characterized from yeast, Drosophila, Xenopus, and human sources. Yeast SWI/ SNF consists of 11 identified subunits, including a highly conserved ATPase, Swi2/Snf2. A major consequence of SWI/SNF action is the increased accessibility of transcription factors to nucleosomal DNA (10). The complex is able to catalyze the movement of histones along DNA in cis, as well as the displacement of histones from DNA in trans (47,51,68). Recently, it was shown that SWI/SNF generates superhelical torsion within linear DNA fragments and changes in DNA twist, providing a means for the repositioning of nucleosomes along DNA (17,24). Yeast SWI/SNF is able to bind to both DNA and nucleosomes; however, it does so without sequence specificity (9, 52). DNA expression microarray analysis has shown that levels of only a subset (ca. 5%) of yeast genes are affected by the loss of SWI/SNF ATPase activity, including the acid phosphatase and mating-type specific genes (25, 56). Many recent studies have indicated that yeast SWI/SNF and its human counterparts are able to interact with sequence-specific transcription factors, which may recruit the complex to specific genes. A human SWI/SNF complex, E-RC1, functionally and physically interacts with erythroid Krüppel-like factor (EKLF) to increase transcription of the ␤-globin gene (2, 27, 36), and C/EBP␤ interacts with hSWI/SNF to activate myeloid genes (33). Recently, BRG1-containing hSWI/SNF was shown to associate with human heat shock factor 1 (hHSF1) (57). Interactions between SWI/SNF and the glucocorticoid receptor have also been reported (16,45,65,73).Work from our lab and others has shown that purified yeast SWI/S...

We analyzed the targeting of histone acetyltransferase (HAT) complexes by DNA-binding activators during transcriptional activation and the resulting distribution of acetylated histones. An in vitro competition assay was developed to acetylate and transcribe a nucleosomal array template in the presence of excess non-speci®c chromatin, which mimics in vivo conditions. Stimulation of transcription from the nucleosomal array template under competitive conditions by the SAGA and NuA4 HAT complexes depended on the presence of the Gal4-VP16 activator, which recognizes sites in the promoter and directly interacts with these HATs. Importantly, the stimulation of transcription by SAGA and NuA4 depended on the presence of Gal4-VP16 during histone acetylation, and Gal4-VP16-bound nucleosomal templates were acetylated preferentially by SAGA and NuA4 relative to the competitor chromatin. While targeting of the SAGA complex led to H3 acetylation of promoter-proximal nucleosomes, targeting of the NuA4 complex led to a broader domain of H4 acetylation of >3 kbp. Thus, either promoter-proximal H3 acetylation by SAGA or broadly distributed acetylation of H4 by NuA4 activated transcription from chromatin templates.

The SWI/SNF complex is required for the expression of many yeast genes. Previous studies have implicated DNA binding transcription activators in targeting SWI/SNF to UASs and promoters. To determine how activators interact with the complex and to examine the importance of these interactions, relative to other potential targeting mechanisms, for SWI/SNF function, we sought to identify and mutate the activator-interaction domains in the complex. Here we show that the N-terminal domain of Snf5 and the second quarter of Swi1 are sites of activation domain contact. Deletion of both of these domains left the SWI/SNF complex intact but impaired its ability to bind activation domains. Importantly, while deletion of either domain alone had minor phenotypic effect, deletion of both resulted in strong SWI/SNF related phenotypes. Thus, two distinct activator-interaction domains play overlapping roles in the targeting activity of SWI/SNF, which is essential for its function in vivo.

The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, 1, in both yeast and mammalian cells. GR-1 can directly interact with purified SWI-SNF complex, and mutations in 1 that affect the transactivation activity in vivo also directly affect 1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate 1-driven transcription from chromatin templates in vitro. Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of 1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions.