reflects our work on three different specific aims: 1. TRANSCRIPTIONAL ACTIVATION AND SWI/SNF-DEPENDENT NUCLEOSOME MOBILIZATION. In collaboration with Dr. Bruce H. Howard (NICHD). 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 non-induced and transcriptionally activated chromatin. In the absence of the Gcn4 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 Gcn4 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. We speculate that remodeling entire genes might facilitate elongation through nucleosomes by RNA polymerase II. The advent of high throughput sequencing has allowed us to analyse nucleosome positioning on a genome-wide scale, confirming and extending our previous observations for HIS3. We are using paired-end sequencing, which increases the accuracy of the position measurements and facilitates the bioinformatics analysis. We have evidence for major disruptions in the chromatin structure of genes when they are activated for transcription. We are exploring the roles of remodeling complexes capable of nucleosome mobilization in disruption of chromatin structure. Our collaborator, Dr. Bruce Howard, has written programs to analyze these data. I have written a review on nucleosome positioning: Clark DJ (2010). Nucleosome positioning, nucleosome spacing and the nucleosome code. J Biomol Struc Dyn 27, 781793. 2. A NUCLEOSOMAL BARRIER TO TRANSCRIPTION BY RNA POLYMERASE II IN VITRO AND IN VIVO. In collaboration with the Studitsky Lab (UMDNJ). Many eukaryotic genes are regulated at the level of transcript elongation. Nucleosomes are likely targets for this regulation. Previously, the Studitsky Lab has shown that nucleosomes formed on very strong positioning sequences (601 and 603), present a high, orientation-dependent barrier to transcription by RNA polymerase II in vitro. The existence of this polar barrier correlates with the interaction of a 16-bp polar barrier signal (PBS) with the promoter-distal histone H3-H4 dimer. We find that the polar barrier is relieved by ISW2, an ATP-dependent chromatin remodeler, which translocates the nucleosome over a short distance, such that the PBS no longer interacts with the distal H3-H4 dimer, although it remains within the nucleosome. In vivo, insertion of the 603 positioning sequence into the yeast CUP1 gene results in a modest reduction in transcription, but this reduction is orientation-independent, indicating that the polar barrier can be circumvented. Surprisingly, the 603-nucleosome is present at the expected position in only a small fraction of cells. Thus, the polar barrier is probably non-functional in vivo because the nucleosome is not positioned appropriately, presumably due to nucleosome sliding activities. We suggest that interactions between polar barrier signals and chromatin remodelers might have significant regulatory potential. A manuscript describing this work has been submitted. 3. SPT10 AND SWI4 CONTROL THE TIMING OF HISTONE H2A/H2B GENE ACTIVATION IN BUDDING YEAST. 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) which has been implicated as a global regulator of core promoter activity. We confirmed that Spt10 has global effects on transcription by expression microarray analysis. Surprisingly, we were able to show that the global effects of Spt10 are the indirect result of defects in chromatin structure, reflecting its role as activator of the core histone genes. We demonstrated that Spt10 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. Thus, Spt10 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. The DNA-binding domain of Spt10 contains an unusual zinc finger with homology to foamy retrovirus integrase, which we propose to be a sequence-specific DNA-binding protein. Spt10 is 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 are now exploring the role of Spt10 in the cell cycle-dependent regulation of the histone genes that is necessary to provide histones for nucleosome assembly during DNA replication. In budding yeast, histones H2A and H2B are expressed from divergent promoters at the HTA1-HTB1 and HTA2-HTB2 loci. We show that Spt10 binds to two pairs of UAS elements in the HTA1-HTB1 promoter: UAS1/UAS2 drive HTA1 expression and UAS3/UAS4 drive HTB1. UAS3 and UAS4 also contain binding sites for the cell cycle regulator SBF (a Swi4-Swi6 heterodimer), which overlap the Spt10 binding sites. Spt10 and SBF binding to UAS3 and UAS4 is mutually exclusive in vitro. Both SBF and Spt10 are bound in cells arrested with alpha-factor, apparently awaiting a signal to activate transcription. Soon after removal of alpha-factor, SBF initiates a small, early peak of HTA1 and HTB1 transcription, which is followed by a much larger peak due to Spt10. Both activators dissociate from the HTA1-HTB1 promoter after expression has been activated. Thus, SBF and Spt10 cooperate to control the timing of HTA1-HTB1 expression. A manuscript describing this work has been submitted. Our current work is focused on the identification of negative regulatory proteins that bind at the histone promoters, which might counteract activation by Spt10p. We have identified candidates and are currently validating them.
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