A Mechanism for Coordinating Chromatin Modification and Preinitiation Complex Assembly  Joshua C. Black, Janet E. Choi, Sarah R. Lombardo, Michael Carey  Molecular Cell  Volume 23, Issue 6, Pages 809-818 (September 2006) DOI: 10.1016/j.molcel.2006.07.018 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Immobilized Chromatin Template (A) The template bears five GAL4 binding sites upstream of the adenovirus E4 TATA box (G5E4T) flanked with nucleosome positioning sequences from the sea urchin 5S rRNA gene-separated EcoRI sites (RI). (B) Coomassie blue-stained SDS gel of recombinant octamers following gel filtration. Note that H2A and H2B comigrate in this gel system. (C) Validation of chromatin. Decreasing amounts of naked DNA (100%, 250 ng; 30%, 15%, 5%, and 2.5%), free chromatin (C), and chromatin immobilized on magnetic bead (CIT) were digested with EcoRI. The positions of naked 5S and G5E4T DNA, and mobility shifts induced by nucleosome occupancy, are indicated in the figure. (D) MNase analysis of chromatin. 32P-labeled, immobilized chromatin (250 ng) was subjected to MNase digestion for 10 min at room temperature with from 3 × 10−5 to 3 × 10−3 units of MNase. Molecular Cell 2006 23, 809-818DOI: (10.1016/j.molcel.2006.07.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 GAL4-VP16-Directed Ordered Recruitment of Cofactors to Immobilized DNA and Chromatin (A) Schematic of immobilized template assay timeline. A 30 min activator binding period was followed by incubation with HeLa nuclear extract (NE) for 3, 10, or 30 min. A control reaction was performed in the absence of GAL4-VP16 for 30 min. (B) Immunoblots of proteins bound to immobilized DNA templates after incubation with HeLa NE. (C) Immunoblots of proteins bound to immobilized chromatin templates. (D) In vitro transcription occurs after PIC assembly is complete. DNA templates were incubated with HeLa NE as in (B), except that NTPs were added with the NE and transcription was allowed to proceed for the time indicated. Molecular Cell 2006 23, 809-818DOI: (10.1016/j.molcel.2006.07.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Mediator and p300 Cooperatively Bind DNA (A) Silver-stained gel of affinity purified human Mediator. (B) Silver-stained gel of purified His6-tagged p300. Autoradiograms show that His6-tagged p300 is functional for histone acetylation and autoacetylation in the presence of 3H-acetyl-CoA. (C) Cooperative binding of Mediator and p300 to immobilized templates. We show data only for MED23 subunit of Mediator; however, Mediator binding was also measured by blotting MED1, MED25, and CDK8 in all of our experiments and was consistent with MED23 (data not shown). (D) Cooperative binding of Mediator and p300 results in cooperative HAT activity. Purified Mediator, recombinant p300, and 3H-acetyl-CoA were coincubated with immobilized chromatin templates. Molecular Cell 2006 23, 809-818DOI: (10.1016/j.molcel.2006.07.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Acetylation Dissociates p300 from GAL4-VP16-Mediator Complexes (A) Acetyl-CoA induces p300 dissociation from GAL4-VP16-Mediator. GAL4-V16-Mediator-p300 complexes were assembled on immobilized DNA templates as in Figure 3 (time 0). Complexes were incubated with or without 10 μM acetyl-CoA for the indicated times. (B) Acetylated p300 has significantly reduced affinity for GAL4-VP16-Mediator. p300 was preacetylated by incubation with 10 μM cold ac-CoA at 30°C (ac-p300). Increasing concentrations (7.5, 30, and 120 ng) of mock-treated p300 or preacetylated p300 (ac-p300) were incubated with GAL4-VP16 and Mediator on immobilized DNA templates. Autoacetylation of p300 was measured by immunoblot with an anti-acetyl lysine antibody (right panel). (C) Ac-p300 does not acetylate immobilized chromatin. (D) Ac-p300 retains HAT activity. Molecular Cell 2006 23, 809-818DOI: (10.1016/j.molcel.2006.07.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Autoacetylation Induces a Conformational Change in the p300 HAT Domain (A) ATM-p300 does not autoacetylate. (B) Curcumin inhibits p300 HAT and autoacetylation activities. A HAT assay was performed using p300 with recombinant octamers in the presence of 3H-acetyl-CoA. (C) ATM-p300 does not dissociate in the presence of ac-CoA, but binding to immobilized template is blocked by Curcumin. (D) Curcumin prevents p300 from binding to GAL4-VP16-Mediator on immobilized template. Three hundred micromolar Curcumin or DMSO (Curcumin solvent) was preincubated with p300 where indicated. (E) Protease footprinting of p300, ac-p300, and Curcumin. Mock-treated, acetylated p300 (ac-p300), and Curcumin-treated p300 were incubated with Proteinase K (PK). Cleavage products at different intervals (45, 90, 180, and 240 s) were analyzed by immunoblotting. A plus sign indicates regions hypersensitive to PK when compared to untreated p300, while a minus sign indicates regions protected. Molecular Cell 2006 23, 809-818DOI: (10.1016/j.molcel.2006.07.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Dissociation of p300 by Autoacetylation or Competition with TFIID Is Required for Transcription (A) TFIID competes with p300 for Mediator, which facilitates acetyl-CoA-dependent dissociation of p300 in an immobilized template assay. Purified Mediator (90 ng), recombinant p300 (90 ng), TFIIA (12 ng), or TFIID (1× = 30 ng, 5× = 150 ng) with or without 10 μM acetyl-CoA was incubated with immobilized chromatin templates as indicated. All panels are from the same experiment. (B) In vitro transcription. DNA was preincubated with GAL4-VP16, p300, ac-p300, mock-acetylated ATM-p300, Mediator, and TFIID (1 = 30 ng, 3 = 90 ng, and 10 = 300 ng), as indicated. (C) Timeline of chromatin transcription. (D) In vitro chromatin transcription. Following activator binding, acetylation, and remodeling, repression by ATM-p300 was assayed with the indicated cofactors: ATM-p300, ac-p300, TFIID, or Curcumin (300 μM). HeLa nuclear extract and NTPs were then added to initiate transcription. Molecular Cell 2006 23, 809-818DOI: (10.1016/j.molcel.2006.07.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 ChIP Demonstrates p300 Dissociates prior to Accumulation of Transcript, while VP16 Remains Bound (A) ChIP of U2OS Tet-On cells induced with 1 μg/ml Dox for the indicated times. Immunoprecipitated DNA was analyzed by multiplex PCR with primers to the Tet-On luciferase promoter (Luc) or GAPDH promoter (GAP). Input corresponds to 3% of input chromatin. (B) RT-qPCR analysis of luciferase transcript level. RNA was isolated from U2OS Tet-On cells treated with Dox for the indicated times. cDNA levels of luciferase were normalized to β-actin at each time point, and fold induction was calculated compared to the uninduced samples. Error bars represent the standard deviation. (C) Model for transition from chromatin modification to PIC assembly. Initially, Mediator interacts with and recruits chromatin-remodeling complexes (i.e., p300). Following acetylation, p300 undergoes a conformational change and dissociates. Competition between TFIID and p300 for Mediator facilitates the dissociation. TFIID-Mediator then functions as a scaffold for assembly of GTFs and Pol II, allowing completion of PIC assembly. Molecular Cell 2006 23, 809-818DOI: (10.1016/j.molcel.2006.07.018) Copyright © 2006 Elsevier Inc. Terms and Conditions