Abstract
To investigate the role of chromatin structure in the regulation of transcription by RNA polymerase II, we developed a chromatin transcription system in which periodic nucleosome arrays are assembled with purified recombinant ATP-utilizing chromatin assembly and remodeling factor (ACF), purified recombinant nucleosome assembly protein 1 (dNAP1), purified native core histones, plasmid DNA, and ATP. With this chromatin, we observed robust activation of transcription with three different transcription factor sets (nuclear factor kappaB p65 + Sp1, estrogen receptor, and Gal4-VP16) added either before or after chromatin assembly. In fact, the efficiency of activated transcription from the ACF + dNAP1-assembled chromatin was observed to be comparable with that from naked DNA templates or chromatin assembled with a crude Drosophila extract (S190). With ACF + dNAP1-assembled chromatin, we found that transcriptional activation is dependent upon acetyl-CoA. This effect was not seen with naked DNA templates or with crude S190-assembled chromatin. We further determined that acetyl-CoA is required at the time of preinitiation complex assembly but not during assembly of the chromatin template. These findings suggest that there is at least one key acetylation event that is needed to assemble a functional transcription preinitiation complex with a chromatin template.
Highlights
From the ‡Section of Molecular Biology and Center for Molecular Genetics, University of California San Diego, La Jolla, California 92093-0347 and the §Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262
The cloning of the Acf1 subunit of ACF led to the development of a purified chromatin assembly system that consists of defined components: purified recombinant ACF, purified recombinant dNAP1, purified native core histones, plasmid DNA, and ATP [16]
We compared the transcriptional properties of chromatin assembled with purified ACF ϩ dNAP1 to those of chromatin assembled with the crude S190 extract
Summary
To investigate the role of chromatin structure in the regulation of transcription by RNA polymerase II, we developed a chromatin transcription system in which periodic nucleosome arrays are assembled with purified recombinant ATP-utilizing chromatin assembly and remodeling factor (ACF), purified recombinant nucleosome assembly protein 1 (dNAP1), purified native core histones, plasmid DNA, and ATP. The S190 extract has been a reliable source of chromatin assembly activity, we have recently achieved the ATP-dependent assembly of chromatin with purified, recombinant chromatin assembly factors [16] In these reactions, periodic nucleosome arrays are assembled with purified recombinant ACF, purified recombinant dNAP1, purified native core histones, plasmid DNA, and ATP. With this new chromatin transcription system, we have found that the presence of acetyl-CoA at the time of transcription preinitiation complex assembly is essential for activation with chromatin templates
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