The yeast SPT10 gene encodes a putative histone acetyltransferase (HAT) implicated as a global transcription regulator acting through basal promoters. Here we address the mechanism of this global regulation. Although microarray analysis confirmed that Spt10p is a global regulator, Spt10p was not detected at any of the most strongly affected genes in vivo. In contrast, the presence of Spt10p at the core histone gene promoters in vivo was confirmed. Since Spt10p activates the core histone genes, a shortage of histones could occur in spt10⌬ cells, resulting in defective chromatin structure and a consequent activation of basal promoters. Consistent with this hypothesis, the spt10⌬ phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10⌬ cells, as shown by irregular nucleosome spacing and reduced negative supercoiling of the endogenous 2m plasmid. Chromatin structure plays an essential role in gene regulation. The structural unit of chromatin is the nucleosome, which is composed of 147 bp of DNA wrapped in a negative superhelix around a central octamer of core histones (composed of two molecules each of H2A, H2B, H3, and H4) (25). Nucleosomes are separated by linker DNA, forming a "beads on a string" structure. A fifth histone, H1, binds to both the nucleosome and the linker DNA to drive the coiling of the nucleosomal filament to form the 30-nm fiber.The nucleosome presents a problem for regulatory proteins seeking access to DNA because so much of the DNA is protected by histones: the inner surface of the DNA is completely occluded by the central core, the external surface is at least partly protected by the core histone tail domains, and the DNA coils are so close together that their apposed surfaces are also unavailable. To cope with the intrinsically repressive nature of the nucleosome structure, regulatory proteins recruit two types of chromatin remodeling complex to promoters: (i) chromatin remodeling machines, which use ATP to move nucleosomes, effect nucleosomal conformational changes, and exchange core histones with variants (33); and (ii) chromatin modifying enzymes, which catalyze posttranslational modifications of the histones, mostly in their tail domains. These modifications are proposed to represent a "histone code" which is read by regulatory proteins that recognize particular combinations of modifications, resulting in activation or silencing of chromatin (41). Histone acetylation is generally associated with gene activation and is catalyzed by histone acetyltransferases (HATs). The identification of the Gcn5p coactivator as a HAT led to a breakthrough in the field, connecting transcription factors with chromatin (3). The current paradigm is that histone modifying complexes are cofactors recruited to promoters by sequencespecific activators or repressors.Our studies have focused on the CUP1 gene of Saccharomyces cerevisiae as a model for the role of chromatin in gene regulation (35)(36)(37). CUP1 encodes a metallothionein responsible for protecting cell...

The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p. An episome containing transcriptionally active or inactive CUP1 was purified in its native chromatin structure from yeast cells. The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo. Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes. The chromatin structure of an episome containing inactive CUP1 isolated from ace1⌬ cells is organized into clusters of overlapping nucleosome positions separated by linkers. Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p. Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome. Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p. These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

The relationship between chromatin remodeling and histone acetylation at the yeast CUP1 gene was addressed. CUP1 encodes a metallothionein required for cell growth at high copper concentrations. Induction of CUP1 with copper resulted in targeted acetylation of both H3 and H4 at the CUP1 promoter. Nucleosomes containing upstream activating sequences and sequences farther upstream were the targets for H3 acetylation. Targeted acetylation of H3 and H4 required the transcriptional activator (Ace1p) and the TATA boxes, suggesting that targeted acetylation occurs when TATA-binding protein binds to the TATA box or at a later stage in initiation. We have shown previously that induction results in nucleosome repositioning over the entire CUP1 gene, which requires Ace1p but not the TATA boxes. Therefore, the movement of nucleosomes occurring on CUP1 induction is independent of targeted acetylation. Targeted acetylation of both H3 and H4 also required the product of the SPT10 gene, which encodes a putative histone acetylase implicated in regulation at core promoters. Disruption of SPT10 was lethal at high copper concentrations and correlated with slower induction and reduced maximum levels of CUP1 mRNA. These observations constitute evidence for a novel mechanism of chromatin activation at CUP1, with a major role for the TATA box.Eukaryotic DNA is packaged into the cell nucleus in the form of chromatin. The basic structural repeat unit of chromatin is the nucleosome, in which 147 bp of DNA are wrapped in nearly two superhelical turns around a central core histone octamer that is composed of two molecules each of the four core histones H2A, H2B, H3, and H4. A molecule of histone H1 is bound to the nucleosome core and to the linker DNA and directs the folding of the chromatin fiber. The assembly of DNA into nucleosomes represses transcription in vitro, raising the question of how the cell copes with chromatin. It is now clear that chromatin structure is more than just a DNA packaging system: it is intimately connected with events in gene regulation. This is evident from the identification of two classes of chromatin-modifying activities (17,43,48): (i) remodeling complexes, which use the energy of ATP hydrolysis to effect changes in chromatin structure, and (ii) histone modifying complexes, which modify histones posttranslationally (notably, histone acetyltransferases [HATs] and deacetylases

The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His 2 -Cys 2 zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a "blocking" domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10⌬ mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.