reflects our work on three 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. 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. 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. The questions which form the basis of our current work are: (1) What is 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) How general is chromatin domain remodeling in yeast? We are mapping nucleosomes on a genome-wide scale using high-throughput sequencing. 2. A NUCLEOSOMAL BARRIER TO TRANSCRIPTION BY RNA POLYMERASE II IN VITRO AND IN VIVO. In collaboration with the Studitsky Lab (UMDNJ). Synthetic nucleosome positioning sequences such as 601 and 603 have been invaluable for experiments in vitro in which a uniquely positioned nucleosome is required. Previously, the Studitsky Lab has shown that the 603-nucleosome acts as a polar barrier to transcription by RNA polymerase II in vitro. To test whether the 603-sequence is also a polar block to transcription in vivo, we have inserted the 603-sequence between the transcription and translation start sites of the copper-inducible yeast CUP1 gene. We are determining the effects of the 603-sequence on CUP1 transcription and whether the 603-nucleosome is formed as expected in vivo. We expect to gain insight into the role of nucleosomes in the control of transcript elongation by RNA polymerase II. 3. THE YEAST Spt10 PROTEIN CONTAINS A DNA-BINDING DOMAIN FUSED TO A PUTATIVE HISTONE ACETYLASE DOMAIN. We have shown previously that induction of CUP1 by copper results in targeted acetylation of nucleosomes at the CUP1 promoter. 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 that Spt10p has global effects on transcription by expression microarray analysis. We addressed the mechanism of global regulation. Surprisingly, we were unable to detect Spt10p at any of the most strongly affected genes in vivo using the chromatin immunoprecipitation (ChIP) assay. However, we confirmed that Spt10p is present 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)TTCCN6TTCNC), 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 observed 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, rather than to a classical activation domain. Spt10p is therefore a very unusual trans-activator, in which the HAT domain, normally recruited as a co-activator to promoters through an activation domain, is attached directly to a sequence-specific DNA-binding 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 have proposed that this integrase might also be a sequence-specific DNA-binding protein. To test this hypothesis, we have initiated a new project to determine whether human foamy virus (HFV) integrase is indeed a sequence-specific DNA-binding protein, using a SELEX approach. We have addressed the mechanism through which Spt10p recognizes two UAS elements simultaneously. We demonstrated that Spt10p is an elongated dimer and that its N-terminal domain is necessary for dimer formation. The isolated DNA-binding domain is a monomer and binds tightly to a single UAS element, unlike the full length protein dimer, which requires two UAS elements. We propose that the Spt10p dimer undergoes a major conformational change in order to recognize two UAS elements at the same time. Our current work has the following aims: (1) Demonstration of the putative histone/protein acetylase activity of Spt10p. We are using a proteomics approach. (2) Understanding the role of Spt10p in the cell cycle-dependent regulation of the core histone genes. The cell cycle transcription factor SBF is a heterodimer of Swi4p and Swi6p;Swi4p contains the sequence-specific DNA-binding domain. Swi4-binding sites are predicted in the HTA1-HTB1 promoter;these overlap three of the four UAS elements. We are exploring the possibility that SBF and Spt10p compete for binding to the UAS elements. (3) Identification of negative regulatory proteins at the histone promoters, which might counteract activation by Spt10p. We have identified candidates and are currently validating them.
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