Our overall aim is to understand the mechanisms of gene regulation in the context of chromatin structure. The packaging of DNA into chromatin is generally repressive for transcription. 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 that are recruited to promoters as an integral part of the regulatory process: (1) remodeling complexes, which use the energy of ATP hydrolysis to effect changes in chromatin structure; and (2) histone modifying complexes, which modify histones post-translationally (acetylation, methylation and phosphorylation). Much work has been done to address the question of how genes are activated in the context of chromatin structure using reconstituted chromatin, but comparatively little is known about how native chromatin responds to activation. To address this problem, we have invested a considerable amount of time in developing a new model system based on the yeast CUP1 and HIS3 genes. A method to purify native plasmid chromatin from yeast cells has been developed. The structures of purified plasmid chromatin containing CUP1 in its active and inactive states have been elucidated, providing insight into the remodeling and histone modification events occurring during activation of CUP1 chromatin. This year we have made significant progress on both the CUP1 and HIS3 projects. Targeted histone acetylation at CUP1 (Shen et al. (2002) Mol. Cell. Biol. 22, 6406): CUP1 encodes a copper-inducible metallothionein which protects the cell from the toxic effects of copper. Its regulation is very well understood: the transcriptional activator, Ace1p, binds copper ions and then binds to upstream activating sequences in the CUP1 promoter to activate transcription through its activation domain. It was important to choose a gene with a clearly defined signal transduction pathway, because the long term aim is to reconstitute the activation of CUP1 in vitro using native chromatin as substrate and to dissect the mechanism of activation. We have investigated the relationship between nucleosome movement and histone acetylation at CUP1. Copper induction of CUP1 results in targeted acetylation of both histones H3 and H4 at the CUP1 promoter. The nucleosomes targeted for H3 acetylation are those containing upstream activating sequences and sequences farther upstream. Targeted acetylation of H3 and H4 requires Ace1p and, surprisingly, the TATA boxes, suggesting that targeted acetylation occurs when TBP binds to the TATA box, or afterwards. Unlike acetylation, movement of nucleosomes is not restricted to the promoter but occurs over the entire CUP1 gene and does not require the TATA boxes. This indicates that remodeling is not dependent on targeted acetylation. Targeted acetylation of both H3 and H4 also requires the product of the SPT10 gene, which encodes a putative histone acetylase implicated in regulation at core promoters. Disruption of SPT10 is lethal at high copper concentrations and correlates 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. We propose that the function of targeted histone acetylation at CUP1 is to boost subsequent rounds of transcription by facilitating the re-binding of Ace1p. This proposal is supported by our observation that Ace1p binds to acetylated nucleosomes with much higher affinity than to unacetylated nucleosomes. Remodeling of HIS3 chromatin (Kim and Clark, submitted) We are also working on the yeast HIS3 gene, even though its regulation is more complex than that of CUP1, because the remodeling activity and the histone acetylases acting at HIS3 have been identified. HIS3 is regulated by the Gcn4p transcriptional activator, the Gcn5p histone acetylase and the SWI/SNF remodeling complex. We investigated the remodeling events occurring in a small plasmid containing HIS3, by purifying plasmid chromatin from induced and uninduced cells. Induced chromatin exhibits a reversible, dramatic loss of nucleosomal negative supercoils, which requires Gcn4p and SWI/SNF, but not Gcn5p. Induced chromatin is more accessible to restriction enzymes at the HIS3 promoter, and at other sites far from the promoter. These observations indicate that a chromatin domain containing the entire HIS3 gene is remodeled in a SWI/SNF-dependent process. Remodeling is apparent only after chromatin purification, suggesting that in vivo, supercoils released during remodeling are protected from relaxation and stored within the chromatin structure. Future Work Future work will address the question of the order of recruitment of activator, remodelling activity, histone acetylase, TBP and RNA polymerase II at the CUP1 promoter. This will result in a very detailed picture of activation events at the CUP1 promoter, when combined with our previous data on chromatin structure changes. We will study the structure and function of Spt10p. New HIS3 projects are aimed at understanding the structural basis of the remodelling occurring on induction.
