CREB-binding proteinyp300 are transcriptional coactivators of p65 (nuclear factor-kBycoactivatoryE-selectin)

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-kB (NF-kB) 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 Nand C-terminal domains of both CBPyp300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBPyp300 with p65 were demonstrated by glutathione Stransferase fusion protein binding, and coimmunoprecipitationyWestern 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. Transcriptional regulation, a critical control mechanism in fundamental biologic processes, requires the participation of several classes of proteins: those that bind specific DNA sequences, those that associate with transcriptional regulators through protein–protein interactions, known as transcriptional coactivators or corepressors, and those that perform an architectural function. Collectively, these proteins interact with components of the basal transcriptional apparatus to effect dramatic changes in gene expression (for review, see ref. 1). Nuclear factor-kB (NF-kB) encompasses an important family of inducible transcriptional activators, critical in the regulation of gene expression in response to injury and inflammatory stimuli. The family members include p50, p52, p65 (RelA), c-Rel, and RelB. In the cell, NF-kB exists as homoor heterodimers with distinct DNA binding specificities (for review, see refs. 2–7). The family members share a 300-aa Rel homology region that is responsible for protein dimerization, nuclear localization, and DNA binding to kB elements in the enhancer regions of target genes. A heterodimer composed of a p50 subunit bound to a p65 (Rel A) subunit is the common dimer. The p65 subunit, similar to two others in the kB family, RelB and c-Rel, contains two transactivation domains in the C-terminal region of the protein (8). The nuclear localization signal of NF-kB is masked by the binding of an inhibitory protein, IkB, sequestering NF-kB in the cytoplasm in unstimulated cells. Cellular stimulation with inflammatory cytokines, phorbol esters, UV irradiation, or potent oxidants results in the phosphorylation, ubiquitination, and proteosomal degradation of IkB (9–12). This is followed by the rapid translocation of NF-kB to the nucleus where it binds to specific kB elements (13, 14). Many genes involved in the inflammatory response are induced by NF-kB including the proinflammatory cytokines [interleukin (IL) 1, tumor necrosis factor a (TNF-a), granulocyte–macrophage colony-stimulating factor, IL-2, IL-11, and IL-17], chemokines (IL-8, RANTES, and MCP-1); monocyte chemotactic protein-1 enzymes (inducible nitric oxide synthase and cyclooxygenase II), and the endothelial–leukocyte adhesion molecules [intercellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin]. Mice or cell lines derived from mice deficient in p50 (15), p65 (16), RelB (17), c-Rel (18), and IkB-a (19, 20) display various functional immune response defects, emphasizing the critical importance of NF-kB in inflammation. A second class of proteins important in the initiation of transcription by RNA polymerase II are the coactivators (i.e., proteins that bridge the transcriptional activators and the components of the basal transcriptional apparatus). CREB-binding protein (CBP) is a coactivator that interacts with the cAMPresponse element binding protein (CREB), a process dependent on the cAMP-dependent protein kinase A and its phosphorylation of CREB (21, 22). A closely related cofactor, p300, was independently isolated on the basis of its interactions with adenovirus E1A (23). Both CBP and p300 interact with a variety of transcriptional activators, including CREByATF (22, 24, 25), c-Jun (25, 26), c-Myb (27), YY1 (28),Myo-D (29), c-Fos (30), and steroid receptors (31, 32) (for review, see ref. 33). The interactions between the transcriptional activator and coactivator can be phosphorylation-dependent (e.g., CREB) or -independent (e.g., c-Jun, YY1, or c-Myb) (25, 28, 29). CBPyp300 also bind to the basal transcription factor TFIIB, which in turn contacts the TATA box binding protein (TBP) of the TFIID complex in the basal apparatus (22, 25), as well as an associated H3 and H4 histone acetylating enzyme, p300yCBP-associated factor (Py CAF) (34). CBPyp300 coactivators may perform an important role in the integration of diverse signaling pathways that result in changes in gene expression. For example, competition for limiting amounts of CBPyp300 by different transcription factors activated by diverse signaling pathways may allow specific cellular responses to appropriate signals (31). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y942927-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: ATF-2, activating transcription factor 2; CBP, CREBbinding protein; CREB, cAMP response element binding protein; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; VCAM-1, vascular cell adhesion molecule 1; TNF-a, tumor necrosis factor a; HUVEC, human umbilical vein endothelial cell; TBP, TATA box binding protein; IL, interleukin; PD, positive regulatory domain; HMG Y(I), high mobility group protein I(Y). §To whom reprint requests should be addressed at: Department of Pathology, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, MA 02115. e-mail: [email protected].