Gene activation involves the regulated recruitment of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II and subsequent transcript elongation. These events must occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent the chromatin block, eukaryotic cells possess a set of chromatin remodeling and nucleosome modifying complexes. The former (e.g. the SWI/SNF complex) use ATP to drive conformational changes in nucleosomes and to slide nucleosomes along DNA. The latter contain enzymatic activities (e.g., histone acetylases) which modify the histones post-translationally to mark them for recognition by other complexes. Geneticists have described many interesting connections between chromatin components and transcription, but a system to investigate the structural basis of these has been lacking. We have developed such a model system, involving native plasmid chromatin purified from the yeast Saccharomyces cerevisiae, to perform high resolution studies of the chromatin structures of active and inactive genes. Remarkably, they reveal that activation correlates with large scale movements of nucleosomes and conformational changes within nucleosomes over entire genes. Current work is focused on: (1) understanding the role of this extensive remodeling in transcription, and (2) investigating the role of the Spt10p histone acetylase in global gene regulation. 1. Remodeling of HIS3 chromatin by the SWI/SNF and Isw1 complexes We chose budding yeast as a model organism because biochemical studies of chromatin structure could be combined with molecular genetics. Current models for the role of the SWI/SNF ATP-dependent chromatin remodeling complex in gene regulation are focused on promoters, where the most obvious changes in chromatin structure occur. However, using our plasmid model system with yeast HIS3, a SWI/SNF-regulated gene, we discovered that induction of HIS3 creates a domain of remodeled chromatin structure that extends far beyond the promoter, to include the entire gene. Induction results in a dramatic loss of nucleosomal supercoiling, a decompaction of the chromatin, and a general increase in the accessibility of the chromatin to restriction enzymes. Formation of this domain requires the SWI/SNF complex and the activator Gcn4p, but not the TATA boxes, indicating that remodeling is not the result of transcription. The implication is that the nucleosomes have been opened up in a SWI/SNF-dependent remodeling reaction. We propose that the SWI/SNF complex is recruited to the HIS3 promoter by Gcn4p and then directs remodeling of a chromatin domain. This might facilitate transcription through nucleosomes, in keeping with a possible role for the SWI/SNF complex as an elongation factor (Kim and Clark, 2002). Analysis of nucleosome positioning on HIS3 using the monomer extension technique (Kim et al., 2004) has established that activation results in the disruption of an ordered nucleosomal array by the sliding of nucleosomes into the linker regions. This mobilization of nucleosomes requires the SWI/SNF complex. The Isw1-remodeling complex is also required for remodeling nucleosome structure. These two remodeling machines have both cooperative and antagonistic roles in the activation of HIS3 chromatin (manuscript in preparation). Our current work on this project has the following aims: (1) Elucidation of the structure of the remodeled nucleosome. There are at least two possibilities: unstable nucleosomes (remodeled such that they fall apart easily) and nucleosomes with a dramatically altered conformation. (2) Determination of the roles of histone modifications in remodeling (i.e., histone acetylation, methylation, phosphorylation and ubiquitination). Our work on CUP1 and HIS3 indicates that, at least for these two genes, the target of remodeling complexes is a domain rather than just the promoter. This is an important finding, because it suggests that remodeling complexes act on chromatin domains. What is the function of domain remodeling? We speculate that remodeling entire genes might facilitate elongation through nucleosomes by RNA polymerase II. In a wider context, the fact that remodeling complexes can participate in the formation of chromatin domains might be important in understanding the formation of domains in higher eukaryotes (Oliver et al., 2002). 2. Global regulation by the yeast Spt10 protein is mediated through chromatin structure and the histone upstream activating sequences (UAS elements). Our studies focused initially on the CUP1 gene of Saccharomyces cerevisiae as a model for the role of chromatin in gene regulation. CUP1 encodes a metallothionein responsible for protecting cells from the toxic effects of copper. We have shown that induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter (Shen et al., 2002). This acetylation is dependent on SPT10, which encodes a putative histone acetyltransferase (HAT) related to Gcn5p. SPT10 is not an essential gene, but the null allele is associated with very slow growth and defects in gene regulation. SPT10 has been implicated as a global regulator of core promoter activity. We have addressed the mechanism of this global regulation (Eriksson et al. 2005): Expression microarray analysis confirmed that Spt10p is indeed a global regulator: more than 800 genes are affected by more than 2-fold (mostly repressed by Spt10p). However, using chromatin immunoprecipitation (ChIP), we were unable to detect Spt10p at any of the most strongly affected genes in vivo, but we were able to detect Spt10p at the core histone gene promoters in vivo. Since Spt10p activates the core histone genes, a shortage of histones could occur in spt10-null cells, resulting in defective chromatin structure and consequent activation of basal promoters. Consistent with this hypothesis, the spt10-null phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10-null cells, as shown by irregular nucleosome spacing and reduced negative supercoiling of endogenous plasmid chromatin. Furthermore, we find that Spt10p binds specifically and highly cooperatively to pairs of UAS elements in the core histone promoters (consensus: (G/A)TTCCNNNNNNTTCNC), consistent with a direct role in histone gene regulation. Spt10p does not bind to the promoters of any of the genes strongly affected in the null mutant, or to the CUP1 promoter. No other high affinity sites are predicted in the yeast genome. Our observations are consistent with the idea that the global changes in gene expression in spt10-null cells are actually the indirect effects of defective regulation of the core histone genes. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain. Our current work has the following aims: (1) Identification of the DNA-binding domain of Spt10p. (2) Demonstration of the putative histone/protein acetylase activity of Spt10p. (3) Identification of proteins which interact with Spt10p using yeast """"""""protein chips"""""""". (4) Investigation of the homology between the zinc fingers of Spt10p and human foamy virus (HFV) integrase.
