1. Volume 1, Issue 2, Pages 277-287 (January 1998)
Recruitment of CBP/p300 by the IFNβ Enhanceosome Is Required for Synergistic Activation of Transcription 
Menie Merika, Amy J. Williams, Guoying Chen, Tucker Collins, Dimitris Thanos 
Molecular Cell 
Volume 1, Issue 2, Pages (January 1998)
DOI: /S (00)

2. Figure 1 Synergistic Transcriptional Activation of the Human IFNβ Gene
Mouse P19 cells were transfected with the wild-type or the helically altered IFNβ CAT reporter constructs (200 ng) along with the indicated IFNβ gene activators expressing plasmids. The amount of CAT activity obtained in the absence of transfected activators was taken as 1. Synergism was defined by dividing the CAT activity obtained in each combination of activators by the sum of CAT activities obtained when the same activators were tested separately. Shown is the average of nine independent experiments. The variability of the absolute levels of transcription from experiment to experiment was less than 40%, whereas the variability between different combinations was less than 20%. The amount of each activator transfected was determined after initial titrations. The highest synergism was obtained by transfection of 50 ng of NF-κB (equimolar mixture of p50- and p65-expressing plasmids), 250 ng of IRF1, and 400 ng of ATF2/c-Jun-expressing plasmids. The total amount of DNA was brought to 6 μg by adding vector DNA.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2
Identification of a Specific Domain in the p65 Subunit of NF-κB Required for Synergistic Stimulation of Transcription
(A) Mouse P19 cells were transfected with a reporter containing a single GAL4 site CAT reporter plasmid (0.5 μg) along with the indicated GAL4-p65-AD-expressing plasmids (200 ng) either alone or with the GAL4-IRF1 AD vector (2000 ng). The total amount of DNA was brought to 6 μg by adding pCDNA3. The transfection cocktail contained 100 ng of CMV-βGAL plasmid used as an internal transfection efficiency control.
(B) P19 cells were cotransfected with the IFNβ CAT reporter construct (200 ng) along with the p50/p65 derivatives (50 ng), IRF1 (250 ng), and ATF2/c-Jun (400 ng) expression vectors (indicated at the left). Synergism is as defined in Figure 1.
(C) Same as in (B) but the transfection cocktail did not contain IRF1- and ATF2/c-Jun-expressing plasmids. The variability was less than 15%. (SD) and (AD) depict the synergism and typical activation domains of p65.
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 3
CBP Potentiates the Transcriptional Activity of the IFNβ Gene Enhanceosome
(A) The amino terminal 771 aa of CBP contact the IFNβ activators. Shown is a GST protein–protein interaction experiment using in vitro translated and 35S-labeled IRF1 (lanes 1–3), ATF2 (lanes 4–6), p65 (lanes 7–9), and c-Jun (lanes 10–12) incubated with glutathione beads–immobilized GST-CBP(1–771) (lanes 1, 4, 7, and 9), GST-CBP(706–1069) (lanes 2, 5, 8, and 11), or GST alone (lanes 3, 6, 9, and 12). The GST-bound proteins were analyzed by PAGE and detected by autoradiography. The input (1/5) amount used in these interactions is shown at the left of the figure.
(B) CBP potentiates virus-induced IFNβ transcription. COS cells were cotransfected with the reporter plasmids (2 μg) indicated at the bottom of the figure along with 6 μg of a CBP-expressing vector. The CAT activities derived from mock or Sendai virus–induced extracts were determined as detailed in Figure 3C. Shown is the average of 15 independent experiments. The fold stimulation of virus-dependent transcription from the wild-type enhancer by CBP varied from 2- to 11-fold. However, the variability between individual constructs was less than 10%.
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 4
The Synergism-Specific Domain of p65 Functions by Recruiting CBP to the Enhanceosome
(A) High-affinity binding of CBP with p65 requires the synergism-specific domain. Shown are the results of protein–protein interaction experiment, using glutathione beads–immobilized GST-CBP(1–771) (lanes 1, 4, 7, 10, 13, 16, and 19), GST-CBP(706–1069) (lanes 2, 5, 8, 11, 14, 17, and 20), or GST alone (lanes 3, 6, 9, 12, 15, 18, and 21) and the indicated in vitro translated 35S-labeled p65 derivatives. The input amount (1/5) used in the interactions is shown at the left of the figure.
(B) The synergism-specific domain is required for recruitment of CBP by the enhanceosome. COS cells were cotransfected with the IFNβ CAT reporter (100 ng) along with the indicated activators (300 ng of IRF1, 500 ng of ATF2/c-Jun, and 100 ng p50/p65 derivatives expressing plasmids in the presence or the absence of 3 μg CBP-expressing vector. Shown is the average of seven independent experiments, and the variability was less than 30%.
(C) COS cells were transfected with 1 μg of IFNβ CAT reporter plasmid along with 0.5 μg of the indicated p50/p65 derivatives. Line 1 shows the amount of virus-induced transcription obtained from the endogenous activators (depicted in line 1). Shown is the average of four independent experiments, and the variability was less than 10%. (SD) and (AD) depict the synergism and typical activation domains of p65.
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 5 The IFNβ Enhanceosome Recruits CBP In Vitro
(A) The amino-terminal 771 aa of CBP are recruited to the DNA by the IFNβ gene activators. Shown is an EMSA experiment using recombinant IFNβ gene activators bound to the IFNβ gene enhancer (−110 to −37) either alone or in the presence of different GST-CBP derivatives as indicated in the figure. The ternary complex between each activator and GST-CBP(1–771) is indicated by the arrows. In some experiments, the GST-CBP(1842–2441) protein can form a weak ternary complex with IRF1 (indicated by a dot in lane 4). The probe was run out of the gel to facilitate resolution of the complexes.
(B) Shown is an EMSA experiment using enhanceosomes assembled with either full-length or truncated IFNβ gene activators and the IFNβ enhancer as a probe. Lanes 1–5 depict DNA binding by each protein alone as indicated on the top of the figure. The amounts of proteins used were 10 ng for IRF1 FL, 3 ng of p50/p65FL, 40 ng of ATF2/c-Jun, 2 ng of IRF1 DBD (aa 1–158), and 0.5 ng of p50/p65 RHR. Lane 6 shows the assembled IFNβ enhanceosome when the same amounts of wild-type IRF1 (IRF1 FL), p50/p65(FL), and ATF2/c-Jun were mixed with 100 ng of recombinant HMG I. Lanes 7–9: Increasing amounts of GST-CBP(1–771) (100, 200, and 400 ng) were added to the preassembled wild-type enhanceosome. Lanes 12–16: Increasing amounts (200 and 400 ng) of GST-CBP(706–1069) (lanes 12 and 13) or GST-CBP(1842–2441) (lanes 15 and 16) were added to preassembled wild-type IFNβ enhanceosomes. Lanes 14–16: An enhanceosome bearing the RHR of p65 instead of the wild protein (lane 14) was incubated with increasing amounts of GST-CBP(1–771) (200 and 400 ng). Lanes 17–19: As in lanes 14–16, but the enhanceosome contains the DNA binding domain of IRF1 and wild-type p65. The ternary complex formed by CBP and the enhanceosome is indicated by the arrow. The probe was run out of the gel to facilitate separation of the complexes.
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 6
Recruitment of CBP to the Enhanceosome by a Novel Surface Generated by the Activation Domains
COS cells were cotransfected with the IFNβ CAT reporter plasmid and the depicted chimeric activators either alone or in combination in the presence or in the absence of the CBP-expressing plasmid. The amounts of the wild-type activators are as in Figure 5. The amounts of the chimeric activator expression vectors were optimized in titration experiments, and they were 250 ng for p50/p65-VP16, 150 ng for IRF1-VP16, 100 ng for IRF1-p65 AD, 250 ng for p65-IRF1 AD, and 400 ng for p50/p65-STAT2. Shown is the average of four independent experiments. The variability in CAT activity was less than 35% from experiment to experiment, but the relative variability among different combinations was less than 15%.
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 7
Recruitment of CBP into the Enhanceosome Is Necessary but Not Sufficient for High Levels of Transcription
(A) COS cells were cotransfected with the reporter and the indicated activators (the amounts are as in Figure 5) along with the RHR-CBP-expressing vector (3 μg). Shown is the average of three independent experiments, and the variability was less than 10%.
(B) Lanes 1–3: COS cells were transfected with the IFNβ CAT reporter plasmid along with either the wild-type CBP expression vector (3 μg) or a derivative containing only the amino-terminal 771 aa. Lanes 4–7: The transfection cocktail contained WT E1A 12S or the mutant E1A Δ2–36 bearing a deletion in its CBP interacting domain along with the CBP-expressing vector. CAT activities were determined from mock or virus-induced extracts. Shown is 1 of 2 independent experiments. (SD) and (AD) depict the synergism and typical activation domains of p65.
Molecular Cell 1998 1, DOI: ( /S (00) )

9. Figure 8
A Model for Synergistic Activation of Transcription by the IFNβ Enhanceosome
Virus infection leads to the cooperative assembly of the IFNβ enhanceosome containing NF-κB (p50/p65 heterodimer), IRF1, and ATF2/c-Jun. The high mobility group protein I(Y) that is required for enhanceosome assembly has been omitted from the picture for simplicity. The activation domains of each of the factors are indicated (AD) as distinct ovals. Due to the enhanceosome assembly, regions of the activation domains form a novel surface that constitutes a high-affinity binding site for the amino terminus of CBP as indicated. Recruitment of CBP by the enhanceosome tethers the polII holoenzyme via its interaction with the carboxyl terminus of CBP. Simultaneously, the activation domains of the activators contact the remaining components of the basal apparatus such as TFIID/A/B as indicated by the arrow. Shown is only a fraction of the components recruited and only some of the potential protein–protein interactions that lead to the assembly of the transcriptional machinery. Synergism in transcription results from the simultaneous recruitment of the two distinct subcomplexes, those of the polII holoenzyme and of the TFIID/A/B.
Molecular Cell 1998 1, DOI: ( /S (00) )