FIG. 4.

STAGA functions as a nucleosome-acetylating transcription coactivator on chromatin in vitro. (A) STAGA acetylates nucleosomes. Fluorography of an SDS-PAGE gel containing 1 μg of either purified HeLa core histones (Free) or native nucleosomes (Nuc.) acetylated with M2-purified STAGA (+) or the control M2-purifed fraction (-/c) in the presence of [³H]acetyl-CoA is shown. The presence of equal amounts of free and nucleosomal histones was confirmed by Coomassie staining (not shown). Arrows indicate the position of core histones H3 and H4. H4 acetylation is very weak and can be detected only with longer exposures with free histones. (B) Micrococcal nuclease digestion analysis of G5-MLP chromatin. Shown is an ethidium bromide-stained agarose gel containing a 123-bp DNA ladder (M) and the DNA products of a time course digestion of G5-MLP chromatin with micrococcal nuclease (MNase). (C) Diagram of the transcription reaction illustrating the order of addition of G5-MLP DNA or chromatin (Template), activator (Gal4-VP16), M2-purified STAGA, acetyl-CoA, ATP, nuclear extract (Txn Factors), and nucleoside triphosphates (NTPs). Times are in minutes. (D) Activator-dependent transcription stimulation by STAGA on chromatin in vitro. Autoradiogram of a urea gel containing specific transcripts (arrow) from transcription reactions performed with DNA or chromatin G5-MLP templates in the presence (+) or absence (−) of the indicated components as described for panel C.