| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We have used a purified recombinant chromatin assembly system, including ACF (Acf-1 + ISWI) and NAP-1, to examine the role of histone acetylation in ATP-dependent chromatin remodeling. The binding of a transcriptional activator (Gal4-VP16) to chromatin assembled using this recombinant assembly system dramatically enhances the acetylation of nucleosomal core histones by the histone acetyltransferase p300. This effect requires both the presence of Gal4 binding sites in the template and the VP16 activation domain. Order-of-addition experiments indicate that prior activator-meditated, ATP-dependent chromatin remodeling by ACF is required for the acetylation of nucleosomal histones by p3O0. Thus, chromatin remodeling, which requires a transcriptional activator, ACF and ATP, is an early step in the transcriptional process that regulates subsequent core histone acetylation. Glycerol gradient sedimentation and immunoprecipitation assays demonstrate that the acetylation of histones by p300 faci
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litates the transfer of H2A-H2B from nucleosomes to NAP-1. The results from these biochemical experiments suggest that : (1) transcriptional activators (e.g., Gal4-VP16) and chromatin remodeling complexes (e.g., ACF) induce chromatin remodeling in the absence of histone acetylation, (2) transcriptional activators recruit histone acetyltransferases (e.g., p300) to promoters after chromatin remodeling has occurred, and (3) histone acetylation is important for a step subsequent to chromatin remodeling and results in the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone such as NAP-1. Our results indicate a precise role for histone acetylation, namely to alter the structure of nucleosomes (e.g., facilitate the loss of H2A-H2B dimers) that have been previously remodeled by the action of ATP-dependent chromatin remodeling complexes. Thus, transcription from chromatin templates is ordered and sequential, with precise timing and roles for ATP-dependent chromatin remodeling, subsequent histone acetylation and alterations in nucleosome structure. Less
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