Fig. 1

NAP proteins enhance activated transcription from an immobilized chromatin template. a Schematic depicting the model transcription template: the HTLV-1 promoter/G-less cassette. b Purified recombinant proteins. The indicated proteins used in the experiments described herein were separated by SDS-PAGE and visualized by Coomassie staining. Molecular weight size markers (kDa) (lane 1), X. laevis histone octamer (10 pmol) (lane 2), linker histone H1.0 (20 pmol) (lane 3), pCREB (Ser-133, 20 pmol) (lane 4), Tax (20 pmol) (lane 5); asterisk denotes GroEL that co-purifies with Tax, p300 (10 pmol) (lane 6), GST-hNap1 (20 pmol) (lane 7), and GST-SET/Taf1β (16 pmol) (lane 8). c Schematic representation of the in vitro transcription assay showing the individual, temporally-distinct steps. d NAP-family proteins enhance activated transcription from the chromatin-assembled promoter template. The repressive effect of chromatin was relieved by the addition of purified, recombinant activators (Tax, pCREB, and p300) and Ac-CoA. The indicated purified, recombinant histone chaperone (0.17 [+] or 0.67 μM [++]) was added in the presence of activators (Tax, pCREB and p300), and in the absence or presence of Ac-CoA (lanes 4–15). The amount of hNAP1 that gave the greatest enhancement (0.67 μM) was used in subsequent experiments. The transcript (RNA), recovery standard (RS), and size markers are shown. Solid vertical lines designate where the image (from the same gel) was spliced to remove irrelevant lanes, or portions of the image arranged to simplify presentation of the data. The experiment was performed at least three times, and a representative experiment is shown. For this, and subsequent in vitro transcription reactions, size markers derived from radiolabeled HpaII-digested pBR322 are shown on the left (bp). Prior to RNA isolation, a radiolabeled 622 bp recovery standard (RS) was added to each reaction to monitor transcript recovery. e hNAP1 stimulation of transcription requires activators, Ac-CoA, and chromatin. In vitro transcription assays were performed on chromatin-assembled (upper panel) or naked DNA (lower panel) templates, in the absence or presence of activators, as indicated. f hNAP1 enhances transcription during the PIC assembly step. In vitro transcription reactions, performed in the absence or presence of Ac-CoA and/or hNAP1 (added at the start of the reaction), is shown for reference (lanes 1–4). The effect of hNAP1 addition at the start of each individual step, denoted by color (see panel b) is shown (lanes 5–8). The effect of hNAP1, added at the start of the indicated step and removed from the reaction at the end of the indicated step (see “Results” for details), is shown (lanes 9–12). All reactions were performed in the presence of activators (Tax, pCREB, p300)