Global regulation by the yeast Spt10 protein is mediated through chromatin structure and the histone upstream activating sequence elements.

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 sequence-specific 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 (35-37). CUP1 encodes a metallothionein responsible for protecting cells from the toxic effects of copper. The induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter. This acetylation is dependent on SPT10 (23, 37), which encodes a putative HAT related to Gcn5p (29). SPT10 was originally identified as one of a set of SPT genes, mutations in which suppress insertion mutations due to the Ty1 transposable element (8). The SPT genes encode many proteins important in transcription, including subunits of the SAGA histone modifying complex (12), TATA-binding protein, and histones (44, 47). SPT10 is not an essential gene, but the null allele is associated with very slow growth and defects in gene regulation (7, 27, 28). SPT10 has been implicated as a global regulator of core promoter activity, acting at or near the TATA box (5, 34, 48). Recently, it has been shown that SPT10 is required for cell cycle-specific acetylation of lysine-56 of H3 at the histone gene promoters (46). However, the mechanism by which Spt10p exerts its effects through basal promoters is unclear. Spt10p has also been identified as an activator of the histone genes, which it regulates in conjunction with Spt21p, the Hir corepressor (7, 15, 38), and SWI/SNF (6, 46). In S. cerevisiae, there are four loci encoding the major core histones, each containing a pair of divergent genes transcribed from a central promoter (31). Two of these loci, HHT1-HHF1 and HHT2-HHF2, carry genes for H3 (HHT1 and -2) and H4 (HHF1 and -2) (40); the others, HTA1-HTB1 and HTA2-HTB2, carry genes for H2A (HTA1 and -2) and H2B (HTB1 and -2) (14). Chromatin immunoprecipitation (ChIP) experiments have shown that Spt10p is present at all four core histone promoters in vivo (15, 46). Here we address the mechanism by which Spt10p acts as a global regulator. Using microarray analysis, we identified hundreds of genes affected by Spt10p. However, we could demonstrate the presence of Spt10p only at the histone gene promoters in vivo, suggesting that Spt10p acts indirectly on the other genes through regulation of the histone genes. In support of this hypothesis, we present evidence that chromatin is incorrectly assembled in spt10Δ cells. Remarkably, we found that Spt10p is a sequence-specific DNA-binding protein which binds with a high affinity to the upstream activating sequences (UAS elements) in all four major core histone promoters. Due to the highly cooperative nature of the binding, no other high-affinity sites are predicted in the yeast genome.