Volume 6, Issue 3, Pages 551-561 (September 2000) Activator-Dependent Transcription from Chromatin In Vitro Involving Targeted Histone Acetylation by p300  Tapas K. Kundu, Vikas B. Palhan, Zhengxin Wang, Woojin An, Philip A. Cole, Robert G. Roeder  Molecular Cell  Volume 6, Issue 3, Pages 551-561 (September 2000) DOI: 10.1016/S1097-2765(00)00054-X

Figure 1 Reconstitution of Chromatin Template Using Purified Proteins (A) Schematic of the plasmid template insert containing a central 690 bp region with five GAL4 binding sites upstream of the adenovirus major late core promoter and a G-less cassette and, on both sides, five direct repeats of a 208 bp 5S DNA nucleosome-positioning sequence. (B) Analysis of purified proteins by SDS-PAGE stained with Coomassie blue R-250. Lane 1, 5 μg of purified human core histones; lane 2, 3 μg of rNAP1; and lane 3, 1 μg of rTopoI. (C) DNA supercoiling assay for assembled chromatin. Lane 1, supercoiled DNA used for assembly; lane 2, relaxed DNA after rTopoI treatment of the DNA of lane 1; lane 3, supercoiled DNA isolated by deproteinization of assembled chromatin. (D) MNase digestion analysis. Assembled chromatin was treated with increasing concentrations of MNase. After deproteinization, the resulting DNA was resolved on a 1.5% agarose gel and stained with ethidium bromide. (E) SDS-PAGE (10%) and Coomassie blue R-250 stain of purified full-length human p300. Molecular Cell 2000 6, 551-561DOI: (10.1016/S1097-2765(00)00054-X)

Figure 2 GAL4-VP16-Mediated Transcription of Chromatin Is Dependent upon p300 and Acetyl-CoA (A) Schematic representation of the in vitro transcription protocol. (B and C) Transcription from naked DNA and chromatin templates. Freshly assembled chromatin or histone-free DNA (28 ng) was subjected to the protocol in (A) with or without GAL4-VP16 activator (40 ng), full-length p300 (25 ng), and acetyl-CoA (1.5 μM) as indicated. Molecular Cell 2000 6, 551-561DOI: (10.1016/S1097-2765(00)00054-X)

Figure 3 HAT Activity of p300 Is Required for Transcription Activation (A) Schematic representation of the modified in vitro transcription protocols. (B) Transcription. Nucleosomal (lanes 1–6) or histone-free (lanes 7–8) pG5ML array templates were subjected either to protocol I (lanes 1–5, 7, and 8) or to protocol II of (A) (lane 6). Addition of activator (GAL4-VP16), full-length p300, acetyl-CoA, and the HAT inhibitor Lys-CoA (15 μM) were as indicated. Molecular Cell 2000 6, 551-561DOI: (10.1016/S1097-2765(00)00054-X)

Figure 4 Activator Targets Histone H3 Acetylation by p300 on Chromatin Templates Protein acetylation reactions were done on chromatin templates either with or without the activator (GAL4-VP16) as indicated, and the samples were analyzed on a 8%–16% SDS-PAGE gel, stained by Coomassie blue (A) before fluorography (B). There was a partial loss of material during handling of the plus activator sample (lane 3). BSA, NAP1, and the histone bands are marked. An asterisk marks weak nonspecific proteins present in the NAP1 preparation. Molecular Cell 2000 6, 551-561DOI: (10.1016/S1097-2765(00)00054-X)

Figure 5 GAL4-VP16-Mediated Promoter-Proximal Acetylation of Histones H3 and H4 by p300 pG5ML array chromatin template was incubated with GAL4-VP16 as indicated. In vitro ChIP assays were then performed (Experimental Procedures) either with no antibody (control) or with anti-acetyl-H3 or anti-acetyl-H4 antibodies as indicated, and the purified DNA was slot blotted and probed with either a promoter-proximal or vector probe as indicated. pG5ML array naked DNA (75 ng) served as a hybridization positive control to normalize signals between the two different probes. To monitor efficiency of IP, 10% of the input was blotted for the promoter-probe hybridization. The data shown are representative of three independent experiments. Molecular Cell 2000 6, 551-561DOI: (10.1016/S1097-2765(00)00054-X)

Figure 6 Effect of Different Activation Domains on p300-Driven Transcription Transcription from DNA (A) or chromatin (B) templates according to the protocol in Figure 2A. GAL4-VP16, GAL4-SP1, and GAL4-CTF1 were added in amounts (40–60 ng) that gave equal activities on DNA templates at saturating levels. Addition of activator, p300, and acetyl-CoA were as indicated. Molecular Cell 2000 6, 551-561DOI: (10.1016/S1097-2765(00)00054-X)

Figure 7 Activation Domains of VP16 and SP1 Interact with Full-Length p300 (A) SDS-PAGE and Coomassie blue R-250 staining analysis of immobilized GST (lane 2), GST-VP16 (lane 3), GST-SP1 (lane 4), and GST-CTF1 (lane 5). All are composed mainly of intact proteins, whereas GAL4-CTF1 shows an intact protein and low molecular weight breakdown products. Lane 1 shows protein standard molecular weight markers (Bio-Rad). (B) Activator interactions with p300 in crude cell lysates. Ten microliters of beads containing 1 μg of GST (lanes 4 and 8) or GST fusion proteins (lanes 5–7 and 9–11) were incubated with Sf21 whole-cell extract containing 100 ng of p300 and analyzed by Western blot with anti-p300 antibodies. Lanes 1–3 show 10%, 5%, and 2.5% of input materials. (C) Activator interactions with purified full-length p300. Ten microliters of beads containing 1 μg of GST (lane 4) or GST fusion proteins (lanes 5–7) were incubated with 100 ng of the purified p300 and analyzed by Western blot. Lanes 1–3 show 5%, 10%, and 20% of input samples. (D) Lack of activator interactions with the p300 HAT domain. Ten microliters of beads containing 1 μg of GST (lane 3) or GST fusion proteins (lanes 4–6) were incubated with 100 ng of the purified p300 HAT domain and analyzed by Western blot. Lanes 1 and 2 show 10% and 20% of input samples. Molecular Cell 2000 6, 551-561DOI: (10.1016/S1097-2765(00)00054-X)