GCN5 Dependence of Chromatin Remodeling and Transcriptional Activation by the GAL4 and VP16 Activation Domains in Budding Yeast

Transcriptional activation in eukaryotes is generally initiated by the binding of an activator to the promoter. Activator binding sites may occur in nucleosome-free regions or may be incorporated into positioned nucleosomes (2, 14, 40, 73, 86). In the latter case, activator binding usually results in nucleosome perturbation (3, 58, 86). We have used yeast episomes having strongly positioned nucleosomes to show that transcriptional activators are capable of perturbing chromatin structure via nucleosomal sites in vivo even outside the context of a natural promoter (46, 66). This perturbation seems likely to result from stable activator binding, although this has not been demonstrated directly, and is strongly dependent on the activation domain (AD) (46, 66, 82). For example, GAL4 perturbs a positioned nucleosome containing its binding site in yeast to a much greater degree in cells grown in galactose medium, when it is in its activating configuration, than in glucose, when it is not, and the chimeric activator GAL4-estrogen receptor (ER)-VP16 similarly is much more effective at this perturbation in the presence of activating β-estradiol than in its absence (66). Consistent with these results, binding of transcription factors is increased by ADs in both yeast and mammalian cells to sites having uncharacterized chromatin structure (9, 70). ADs could facilitate chromatin remodeling via nucleosomal binding sites by contacting components of the preinitiation complex (PIC), such as TFIIB or TATA-binding protein (TBP), and thereby stabilizing activator binding, or by recruiting complexes capable of remodeling or modifying chromatin. Considerable support for activators having both of these functions derives from in vitro and in vivo studies (53, 54, 56). Interactions with components of the PIC could enhance binding through cooperative protein-protein interactions, with additional free energy being provided by nonspecific interactions between PIC components and nearby DNA sequences (12, 72). We have recently shown that fusion of TBP to the DNA-binding domain (DBD) of GAL4 or LexA is not sufficient to remodel via a nucleosomal binding site or to remodel nucleosomes that are near accessible binding sites in vivo (63). However, whether TBP contributes to chromatin remodeling in the context of a true activation domain, or whether other PIC components could be involved in this function, remains to be tested. ADs can recruit chromatin remodeling complexes, which use ATP to alter nucleosome structure in a way that increases accessibility of nucleosomal DNA (54). Recruitment of chromatin-remodeling complexes such as SWI-SNF and NURF can facilitate transcriptional activation of chromatin in vitro, probably by increasing accessibility to PIC components (1, 44, 54). Some genes that undergo chromatin remodeling upon activation in vivo depend on SWI-SNF but others do not, indicating functional redundancy among activities that contribute to chromatin remodeling (16, 22, 28, 30, 45). We have shown that activators can perturb chromatin via nucleosomal sites in swiΔ yeast cells, although some reduction in perturbation of nucleosome positioning was seen for GAL4-ER-VP16 in one case (62). Another potential class of targets for ADs is chromatin-modifying complexes. Chromatin-modifying complexes characterized to date include enzymes, such as GCN5 in the SAGA complex and ESA1 in the NuA4 complex, that can acetylate the amino termini of histones (7). Other histone modifications that may contribute to transcriptional regulation also have been observed and presumably are due to additional complexes (11). GCN5, the prototypical histone acetyltransferase (HAT) (8), contributes to activation of a number of genes in yeast (23, 28, 31, 52, 55, 68). Recruitment of the SAGA complex greatly increases transcriptional activation in vitro (32, 67, 71), and a few examples in which chromatin remodeling depends on GCN5 in vivo have been reported (27, 28, 69). Whether GCN5 contributes to chromatin remodeling by activators via nucleosomal binding sites has not been reported and is the major focus of this paper. Potential redundancy among chromatin-remodeling and -modifying complexes, already alluded to, has been investigated but is not well understood (6, 55, 57, 63, 68). Different activators may differentially recruit distinct complexes, and promoters may vary in their requirements for chromatin remodeling (1, 15, 32, 69). We have attempted to address this issue in this work by comparing chromatin remodeling dependence on GCN5 for ADs varying in type and in strength. We also compare requirements for several ADs at distinct promoters that require chromatin remodeling for their activation.