Transcriptional analysis of chromatin assembled with purified ACF and dNAP1 reveals that acetyl-CoA is required for preinitiation complex assembly

To investigate the role of chromatin structure in the regulation of transcr iption 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 rec ombinant nucleosome assembly protein 1 (dNAP1), purified native core histon es, plasmid DNA, and ATP. With this chromatin, we observed robust activatio n of transcription with three different transcription factor sets (nuclear factor kappaB p65 + Sp1 estrogen receptor, and Ga14-VP16) added either befo re or after chromatin assembly. In fact, the efficiency of activated transc ription from the ACF + dNAP1-assembled chromatin was observed to be compara ble with that from naked DNA templates or chromatin assembled with a crude Drosophila extract (S190). With ACF + dNAP1-assembled chromatin, we found t hat transcriptional activation is dependent upon acetyl-CoA. This effect wa s not seen with naked DNA templates or with crude S190-assembled chromatin. We further determined that acetyl-CoA is required at the time of preinitia tion complex assembly but not during assembly of the chromatin template. Th ese 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.