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. 1997 Apr 1;94(7):2927-32.
doi: 10.1073/pnas.94.7.2927.

CREB-binding protein/p300 are transcriptional coactivators of p65

M E Gerritsen  1 , A J Williams, A S Neish, S Moore, Y Shi, T Collins
Affiliations

CREB-binding protein/p300 are transcriptional coactivators of p65

M E Gerritsen et al. Proc Natl Acad Sci U S A. .

Abstract

CBP (CREB-binding protein) and p300 are versatile coactivators that link transcriptional activators to the basal transcriptional apparatus. In the present study, we identify CBP and p300 as coactivators of the nuclear factor-kappaB (NF-kappaB) component p65 (RelA). Consistent with their role as coactivators, both CBP and p300 potentiated p65-activated transcription of E-selectin and VCAM-1-CAT reporter constructs. The N- and C-terminal domains of both CBP/p300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBP/p300 with p65 were demonstrated by glutathione S-transferase fusion protein binding, and coimmunoprecipitation/Western blot studies. The adenovirus E1A 12S protein, which complexes with CBP and p300, inhibited p65-dependent gene expression. Reporter gene expression could be rescued from E1A inhibition by overexpression of CBP or p300. CBP and p300 act as coactivators of p65-driven gene activation and may play an important role in the cytokine-induced expression of various immune and inflammatory genes.

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Figures

Figure 1
Figure 1
CBP increases transcription of E-selectin (A) and VCAM-1–reporter (B) constructs mediated by p65. COS cells were transfected with 2 μg of reporter gene; 10 μg of the expression plasmid encoding CBP; and 0, 0.1, 0.3, 1, and 3 μg of the expression plasmid encoding p65. Total DNA was kept constant at 12.1 μg by using the empty vector pCR-RSV. The results are representative of three experiments.
Figure 2
Figure 2
E1A inhibits p65-mediated transcription. (A) 12S E1A inhibits p65-mediated transcription of E-selectin reporter gene expression. E-selCAT reporter plasmid (5 μg) and p65 expression plasmids (25 ng) were cotransfected into Schneider cells along with 1, 3, or 10 ng of 12S E1A expression plasmids. (B) 12S E1A inhibits p65-mediated transcription of VCAM-1–CAT reporter gene expression. VCAM-1–CAT reporter plasmid (5 μg) and p65 or Sp1 expression plasmids (25 ng and 5 μg, respectively) were cotransfected into Schneider cells along with 1, 3, or 10 ng of 12S E1A expression plasmids. The results are representative of three experiments.
Figure 3
Figure 3
CBP/p300 and p65 functionally interact in a mammalian two-hybrid system. (A) Structures of the CBP-GAL4 and p65 VP16 constructs. The indicated regions of CBP were cloned into a vector (pM) containing the GAL4 DNA binding domain (BD). Similarly, overlapping regions of p65 were inserted into a vector containing the VP16 activation domain (AD). (B) The N and C termini of CBP interact with the C terminus of p65. COS cells were cotransfected with 2 μg of the pG5CAT reporter gene, 10 μg of the indicated GAL4(pM), and 10 μg of VP16 expression plasmids. Total DNA was kept constant at 22 μg. Data are representative of five experiments. When pooled data were analyzed by the nonparametric Wilcoxon signed rank test, the CBP 1–771 versus CBP 1–771 and p65 286–551 were significantly different (P = 0.015), as were the CBP 1892–2441 versus CBP 1892–2441 and p65 286–551 (P = 0.031). (C) The N and C termini of p300 interact with the C terminus of p65. Cells were cotransfected as described above (B). Data are representative of five experiments. (D) The C terminus of p65 containing the transcriptional activation domain is required for interaction with either the N or C terminus of CBP. Cells were cotransfected as above (B). Data are representative of three experiments.
Figure 4
Figure 4
Physical interaction of CBP/p300 with p65 in vitro and in vivo. (A) Association of CBP/p300 and p65. GST–CBP or GST–p300 fusion constructs were used as ligands and tested for interaction with p65, p50, and Sp1 from programmed COS-cell lysates. Agarose-resin containing GST–CBP [N (amino acids 1–771), M (amino acids 1069–1459), C (amino acids 1892–2441)], or GST–p300 [N (amino acids 1–596), M (amino acids 744-1571), C (amino acids 1572–2370)] were mixed with COS cell lysates. After washing at either high or low salt, bound proteins were released and analyzed by SDS/PAGE on 10% gels followed by Western blot analysis for p65. (B) Physical interaction of CBP/p300 with p65 in endothelial cells. Rows a and b are from TNF-α-treated (100 units/ml, 30 min) HUVECs; row c is from unstimulated HUVECs. Immunoblot with anti-p65 (row a and c) or anti-Sp1 antibody (row b) after immunoprecipitation of whole cell extracts with CBP (lanes 1 and 6), p300 (lane 2), p65 (lanes 3 and 5) antiserum or nonimmune (NI) serum (lane 7). Input, whole cell lysate without immunoprecipitation (lanes 4 and 8).
Figure 5
Figure 5
Model of the TNF-α-induced E-selectin enhancer. After induction by cytokine, heterodimers of NF-κB bind to the promoter, which is constitutively occupied by an ATF-2/c-Jun heterodimer. In parallel with nuclear accumulation of NF-κB, ATF-2 and c-Jun are phosphorylated by p38 kinase and c-Jun N-terminal kinase, respectively (46). The binding of HMG I(Y) at multiple sites increases the binding of NF-κB (39) and bends DNA (49) in a way that facilitates the formation of a higher-order complex. The transcriptional activators make extensive protein–protein contacts with the coactivator and the basal complex. Indicated are interactions between the transactivation region of p65 and the N- and C-terminal regions of CBP, an association between c-Jun and the coactivator, and interactions between TFIIB and CBP. Collectively, these events place multiple transcriptional activators in a favorable architecture to complete for the coactivator that is present in limiting amounts. Because RNA polymerase II is constitutively associated with CBP/p300 (51), binding of the coactivator to the E-selectin enhancer may also efficiently recruit the polymerase.
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