Abstract
Proliferative signals lead to the rapid and transient induction of the c-fos proto-oncogene by targeting the ternary complex assembled on the serum response element (SRE). Transactivation by both components of this complex, serum response factor (SRF) and the ternary complex factor Elk-1, can be potentiated by the coactivator CREB-binding protein (CBP). We report a novel interaction between the bromodomain of CBP, amino acids 1100-1286, and Elk-1. DNA binding and glutathione S-transferase pull-down assays demonstrate that binding requires Elk-1(1-212) but not the C-terminal transactivation domain. Competition and antibody controls show that the bromocomplex involves both SRF and Elk-1 on the c-fos SRE and uniquely Elk-1 on the E74 Ets binding site. Interestingly, methylation interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100-1286) interacts via Elk-1 and does not require specific DNA contacts. Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100-1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters. Repression is relieved moderately or strongly by linking the bromodomain to the N- or C-terminal transactivation domains of CBP, respectively. These results are consistent with a model in which CBP is constitutively bound to the SRE in a higher order complex that would facilitate the rapid transcriptional activation of c-fos by signaling-driven phosphorylation.
Highlights
MAPK1 signaling pathways that play a key role in this response by the activation of immediate early genes like the proto-oncogene c-fos [1]
Our data present evidence for a novel regulatory complex on the c-fos serum response element (SRE). This complex involves the interaction of the bromodomain of CREB-binding protein (CBP) with the ternary complex composed of serum response factor (SRF) and ternary complex factor (TCF)
These interactions seem to be independent of signaling that drives SRE activation. These conclusions are based on the following: 1) The bromodomain of CBP interacts with the C- and N-terminal regions of the TCF Elk-1 in solution
Summary
Materials—Restriction enzymes were obtained from Life Technologies and New England BioLabs. The bromodomain-encoding fragment was recloned into pGEX-2T-6His and clones screened for expression of the GST-bromo fusion protein, as well as into pCDNA3.1-FLAG This vector was used to confirm bromodomain expression by coupled in vitro transcription translation (see below). After three washes with 500 l of RJD*, the beads were collected, resuspended in 20 l of 5ϫ Laemmli buffer (0.25 M Tris-HCl, pH 6.8, 10% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromphenol blue) and denatured for 5 min at 95 °C, and the bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Labeled DNA was purified by organic extraction and alcohol precipitation, resuspended in formamide dye mix, denatured again, and analyzed on 10% sequence gels as described above
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