Interacting Regions in Stat3 and c-Jun That Participate in Cooperative Transcriptional Activation

Clustered specific DNA binding sites for an array of activating transcription factors, plus proteins that bend DNA to facilitate contact between bound proteins, have been documented for a number of vertebrate genes (15, 21, 25, 37). These composite structures have been called enhanceosomes (8). The T-cell receptor alpha (15) and beta-interferon (IFN-β) (25) enhanceosomes, which are assembled in response to dimerization of the T-cell receptor or double-stranded RNA, respectively, have been most thoroughly and profitably explored. Two classes of genes that are very likely dependent on enhanceosome assembly have received a great deal of attention: genes expressed in a tissue-specific manner that acquire multiple binding proteins during development, and genes that are acutely activated by an external stimulus. The latter structures hold appeal for study because they can be examined in cultured cells, in which induced synchronous changes occur in all of the cells under observation, potentially allowing the acute assembly and disassembly of proteins in an enhanceosome to be revealed. The STAT family of transcription factors is activated by the attachment of polypeptide ligands to specific cell surface receptors and, after tyrosine phosphorylation, dimerization, and translocation to the nucleus, can participate within minutes in gene activation (11). It seems likely that STAT molecules bind DNA regions where preenhanceosome structures exist (26, 27) and that the arrival of an activated STAT dimer(s) is the key to forming an active enhanceosome (27). Such a possibility is suggested by experiments showing closely spaced binding sites for STATs and other proteins in the response elements of a number of genes (17, 24, 27, 41). Furthermore, DNase and permanganate treatment of cell nuclei revealed proteins bound at or near Stat1 sites before polypeptide treatment. This was followed by detection of STAT molecules binding close to the same DNA regions after induction (26). One intensively studied set of physiologically important genes that are transcriptionally induced in the liver are the acute-phase response proteins, whose levels increase in the wake of bacterial infections and other toxic assaults. Interleukin-6 (IL-6) stimulation of hepatocytes, via the activation of Stat3, is thought to be the main trigger for inducing the acute-phase genes (18). One of the best-studied enhancers of acute-phase response genes is the α2-macroglobulin enhancer (20) (reviewed in reference 18), a DNA fragment 100 bases long with binding sites for both Stat3 (also called a GAS site) and for AP-1, which includes members of the Fos, Jun, and activating transcription factor (ATF) families of transcription factors. Extracts from liver nuclei of IL-6-treated animals or transformed hepatocytes (hepatoma cells) in culture indicated induction of binding to this region. Since Stat3 and c-Jun interacted in yeast two-hybrid assays and cooperated in maximizing the transcription responses of reporter genes containing the ∼100-bp enhancer (30, 31), it seemed likely that this genomic region would form a STAT-dependent enhanceosome. The experiments presented here were designed to explore this possibility and to uncover the physical basis of c-Jun–Stat3 cooperation. We report evidence, in vitro and in vivo, for an interaction between a region within c-Jun and specific sites within Stat3. Mutations in the proposed contact residues of Stat3 both reduce c-Jun–Stat3 protein interaction and disrupt the cooperation between these two proteins that is required for maximal IL-6-dependent gene activation driven by the α2-macroglobulin enhancer.