| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Gene expression in eukaryotic cells involves the interactions of proteins with DNA packaged into nucleosomes, which are the primary structural units of chromatin, comprising a histone octamer and 147 base pairs of DNA. Since the nucleosome limits the access of trans-acting factors to their cognate DNA sites, nucleosome positioning has been proposed as a mechanism of transcriptional regulation. In this study, we focused roles of nucleosome positioning in regulation of a-cellspecific gene BAR1, an early meiotic gene HOP1, and inducible genes PHO5 and SNZ1. We implemented a new strategy to test the functional significance of the positioned nucleosomes in repression by introducing short, diverse nucleosome-disrupting sequences into a precise genomic region. Increased lengths of poly dA・poly dT and poly d(CG)・poly d(GC) inserts, which can form the B' conformation and Z-DNA, respectively, caused increased expression. Analyses of the genomic chromatin structure revealed that the nucleosome po
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sitions in the promoter regions are destabilized by longer poly dA・poly dT and poly d(CG)・poly d(CG) sequences. These results are consistent with the idea that nucleosome positioning plays an essential role in proper expression of BAR1 and PHO5. We also showed that nucleosome positioning plays important roles in determining promoter accessibility of HOP1 and SNZ1 genes. In addition, we showed that Isw2 and Itc1 are essential components of the Isw2 chromatin remodeling complex to position nucleosomes in BAR1 and HOP1. Finally, we demonstrated that a positioned nucleosome prevents Hap1 activator binding in vivo. These results strongly support the mechanism in which nucleosome positioning has a regulatory function to limit the access of transcription factors. Our results also illustrate that the intrinsic properties of DNA structure make them useful tools for inquiring into local chromatin function in diverse cells and organisms as well as for artificial alteration of gene expression in vivo. Less
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