is divided into two sections, reflecting work on two different specific aims:? ? 1. Transcriptional activation and SWI/SNF-dependent nucleosome mobilization ? ? 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 HIS3, a SWI/SNF-regulated gene, we discovered that transcriptional activation creates a domain of remodeled chromatin structure that extends far beyond the promoter, to include the entire gene. We addressed the effects of transcriptional activation on the chromatin structure of HIS3 by mapping the precise positions of nucleosomes in basal expressing and transcriptionally activated chromatin. In the absence of the Gcn4p activator, the HIS3 gene is organized into a dominant nucleosomal array. In wild type chromatin, this array is disrupted, and several alternative, overlapping nucleosomal arrays are formed. Disruption of the dominant array also requires the SWI/SNF remodeling machine, indicating that the SWI/SNF complex plays an important role in nucleosome mobilization. The Isw1 remodeling complex plays a more subtle role in determining nucleosome positions on HIS3, favoring different positions from those preferred by the SWI/SNF complex. We propose that Gcn4p stimulates nucleosome mobilization over the entire HIS3 gene by the SWI/SNF complex. We suggest that the net effect of interplay among remodeling machines at HIS3 is to create a highly dynamic chromatin structure (Kim et al., 2006). Our work on HIS3 and our earlier work on CUP1 indicate 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. Our current work is aimed at elucidating 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.? ? Kim Y, McLaughlin N, Lindstrom, K, Tsukiyama T, Clark DJ. Activation of Saccharomyces cerevisiae HIS3 results in Gcn4p-dependent, SWI/SNF-dependent mobilisation of nucleosomes over the entire gene. Mol. Cell. Biol. 2006;26:8607-8622.? ? ? 2. The yeast Spt10 protein contains a DNA-binding domain fused to a putative histone acetylase domain? ? We have shown that induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter (Shen et al., 2002; Clark and Shen, 2006). This acetylation is dependent on SPT10, which encodes a putative histone acetylase (HAT). SPT10 has been implicated as a global regulator of core promoter activity. We confirmed this by expression microarray analysis and then addressed the mechanism of global regulation. We were unable to detect Spt10p at any of the most strongly affected genes in vivo using the chromatin immunoprecipitation (ChIP) assay (including CUP1), but we confirmed its presence at the core histone gene promoters, which it activates. We presented evidence that a defective chromatin structure is formed in the absence of Spt10p, with consequent activation of basal promoters. Furthermore, we find that Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequences (UAS elements) in the core histone promoters (consensus: (G/A)TTCC N6 TTCNC), consistent with a direct role in histone gene regulation. 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. We have identified the DNA-binding domain of Spt10p: it contains an unusual zinc finger (His2-Cys2) which has homology to the DNA integrase of foamy retroviruses. We propose that this integrase might also be a sequence-specific DNA-binding protein (Mendiratta et al., 2006). We have also shown that Spt10p is a dimer and that the N-terminal domain is required for dimer formation (Mendiratta et al., 2007). Our current work has the following aims: (1) Demonstration of the putative histone/protein acetylase activity of Spt10p. (2) Identification of proteins which interact with Spt10p. (3) Identifying the molecular mechanisms underpinning the cell cycle regulation of the core histone genes.? We have also initiated a new project to determine whether human foamy virus (HFV) integrase is indeed a sequence-specific DNA-binding protein.? ? Mendiratta G, Eriksson PR, Clark DJ. Cooperative binding of the yeast Spt10p activator to the histone UAS? elements is mediated through an N-terminal dimerisation domain. Nucl. Acids Res. 2007;35:812-821.? ? COLLABORATORS? ? Toshio Tsukiyama, PhD, Fred Hutchinson Cancer Research Center, Seattle, WA.
