Signal transducers and activators of transcription (STAT proteins) bind to palindromic sequence elements related to interferon y (IFN-y) activation sites, which were first identified in the promoters of IFN-'yinducible genes. Although the sequences of the natural palindromic STAT-binding elements vary considerably, they conform to the general structure TT(N)5AA. We have systematically examined the effects of the spacing between the TT and AA core half sites on the binding of the STAT complexes activated by IFN-y, interleukin (IL) 6, granulocytemacrophage colony-stimulating factor, and IL-4. We show that (i) as suggested earlier, a core palindromic TT-AA motifwith a 5-bp spacing displays general STAT binding, (ii) a palindromic motif with a spacing of 4 bp selectively binds to complexes containing Stat3, and (iii) a motif with a 6-bp spacing selectively binds the STAT complexes activated by IL-4. We have examined natural elements in the promoters of cytokine-responsive genes that differ in half-site spacing and found that they display binding properties predicted from the synthetic binding sites. Furthermore, the observed differential selective binding characteristics for the most part correlate with the ability to mediate transcriptional activation of transfected test genes in response to the cytokines tested. Our results thus demonstrate that the specificity of STAT-directed transcription in response to particular cytokines or cytokine families depends in part on the spacing of half sites within the conserved response element sequence.

Transcriptional activation by the HIV-1 Tat protein requires specific residues in the hexanucleotide loop and trinucleotide bulge of the TAR RNA stem-loop structure found in the 5'-untranslated leader of all viral transcripts. Tat directly contacts residue U 22 in the bulge and is thought to act in concert with cellular factors bound to the loop. We find that HeLa nuclear extracts contain two specific TAR RNA-binding proteins, designated TRP-1 and TRP-2, which compete for binding to the upper portion of the TAR hairpin. Analysis of point mutants in TAR RNA reveals that TRP-1 contacts residues in the loop that are important for trans-activation, whereas TRP-2 contacts the bulge, including the same residue (U 22) that is required for the Tat-TAR interaction. Glycerol gradient sedimentation and UV cross-linking experiments indicate that TRP-1 is a large heteromeric complex containing a 185-kD RNA-binding protein, whereas TRP-2 activity derives from a family of 110-to 70-kD proteins. Interestingly, both TRP-1 and TRP-2 promote TAR-dependent transcription in vitro in the presence of Tat, although mixing experiments indicate that each of the three proteins must bind independently to TAR RNA. These findings suggest that the TAR element is recognized by two different nuclear RNA-binding proteins that affect transcriptional regulation by Tat.

Activation of members of the STAT (signal transducers and activators of transcription) family of latent transcription factors is an early event following the binding of many cytokines to their cognate receptors. Although the patterns of STATs activated by different cytokines are well described, the consequences of differential STAT activation are less well studied. We show by mutational analysis that STAT binding elements (SBEs) exist that discriminate between STAT complexes containing STAT1 alpha, STAT3 or both, and that these elements show altered cytokine responsiveness. We also show that in the context of a minimal promoter, single and multiple SBEs exhibit strikingly different patterns of transcriptional activation in response to IFN-gamma, IL-6, OSM or LIF. These differences in transcriptional activation are correlated with the differential ability of these cytokines to activate STAT1 alpha, STAT3 or both. Our results show that the pattern of STATs activated by a cytokine and the arrangement and sequence of the SBEs in the responding promoter have a profound effect on the ability of the cytokine to elicit a transcriptional response.

The STAT (signal transducer and activator of transcription) signaling pathway is activated by a large number of cytokines and growth factors. We sought to design a conditionally active STAT that could not only provide insight into basic questions about STAT function but also serve as a powerful tool to determine the precise biological role of STATs. To this end, we have developed a conditionally active STAT by fusing STATs with the ligand-binding domain of the estrogen receptor (ER). We have demonstrated that the resulting STAT-ER chimeras are estrogen-inducible transcription factors that retain the functional and biochemical characteristics of the cognate wild-type STATs. In addition, these tools have allowed us to evaluate separately the contribution of tyrosine phosphorylation and dimerization to STAT function. We have for the first time provided experimental data supporting the model that the only apparent role of STAT tyrosine phosphorylation is to drive dimerization, as dimerization alone is sufficient to unmask a latent STAT nuclear localization sequence and induce nuclear translocation, sequence-specific DNA binding, and transcriptional activity.The JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway, a recently discovered signaling pathway utilized by many cytokines and growth factors, was first elucidated in the context of interferon (IFN) signaling (11). It was later discovered that a large number of cytokines and growth factors, including most if not all of those that act through the cytokine receptor superfamily, activate overlapping sets of STAT family members, often in addition to activating other signaling pathways (11). IFN-␥ signaling remains, however, a canonical example (2, 56). IFN-␥ binding mediates IFN-␥ receptor chain aggregation, which activates two cytoplasmic tyrosine kinases belonging to the JAK family, Jak1 and Jak2, that associate with the cytoplasmic face of the IFN-␥ receptor chains. Upon receptor oligomerization, the JAKs phosphorylate each other and Tyr 440 of the IFN-␥ receptor ␣ chain. Then Stat1, a latent cytoplasmic transcription factor that is a member of the STAT gene family, is recruited via its Src homology 2 domain (SH2 domain) to the phosphorylated Tyr 440 of the receptor, whereupon Stat1 is itself phosphorylated by the JAKs on a specific tyrosyl residue, Tyr 701. Phosphorylation triggers Stat1 homodimerization via the reciprocal binding of the SH2 domain of one Stat1 monomer with the phosphotyrosyl tail of the other Stat1 monomer in a head-totail interaction. It is thought that phosphorylation is the sole trigger for dimerization. Although it has been hypothesized that dimerization (and not tyrosine phosphorylation per se) in turn triggers nuclear translocation, there are no data that clearly demonstrate this. Indeed, this hypothesis has been challenged by recent studies on Stat5 activation by prolactin, as it has been reported that Stat5 tyrosine phosphorylation and Stat5 nuclear localization are controlled by different pathways that ca...

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