Interleukin-4 (IL-4) stimulation of cells leads to the activation of multiple signaling pathways, one of which involves Stat6. We have generated Stat6-deficient mice by gene targeting in embryonic stem cells to determine the role of this transcription factor in mediating the biologic functions of IL-4. IL-4-induced increases in the cell surface expression of both MHC class II antigens and IL-4 receptor are completely abrogated, and lymphocytes from Stat6-deficient animals fail to proliferate in response to IL-4. Stat6-deficient B cells do not produce IgE following in vivo immunization with anti-IgD. In addition, Stat6-deficient T lymphocytes fail to differentiate into Th2 cells in response to either IL-4 or Il-13. These results demonstrate that, despite the existence of multiple signaling pathways activated by IL-4, Stat6 is essential for mediating responses to IL-4 lymphocytes.

Interleukin-4 (IL-4) is an immunomodulatory cytokine secreted by activated T lymphocytes, basophils, and mast cells. It plays an important role in modulating the balance of T helper (Th) cell subsets, favoring expansion of the Th2 lineage relative to Th1. Imbalance of these T lymphocyte subsets has been implicated in immunological diseases including allergy, inflammation, and autoimmune disease. IL-4 may mediate its biological effects, at least in part, by activating a tyrosine-phosphorylated DNA binding protein. This protein has now been purified and its encoding gene cloned. Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated IL-4 Stat. Study of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the IL-4 receptor provided evidence for direct coupling of receptor and transcription factor during the IL-4 Stat activation cycle. Such observations indicate that IL-4 Stat has the same functional domain for both receptor coupling and dimerization.

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The promoters of a variety of plant genes are characterized by the presence of a G‐box (CCACGTGG) or closely related DNA motifs. These genes often exhibit quite diverse expression characteristics and in many cases the G‐box sequence has been demonstrated to be essential for expression. The G‐box of the Arabidopsis rbcS‐1A gene is bound by a protein, GBF, identified in plant nuclear extracts. Here we report the isolation of three Arabidopsis thaliana cDNA clones encoding GBF proteins referred to as GBF1, GBF2 and GBF3. GBF1 and GBF2 mRNA is present in light and dark grown leaves as well as in roots. In contrast, GBF3 mRNA is found mainly in dark grown leaves and in roots. The deduced amino acid sequences of the three cDNAs indicate that each encodes a basic/leucine zipper protein. In addition, all three proteins are characterized by an N‐terminal proline‐rich domain. Homodimers of the three proteins specifically recognize the G‐box motif, with GBF1 and GBF3 binding symmetrically to this palindromic sequence. In contrast, GBF2 binds to the symmetrical G‐box sequence in such a way that the juxtaposition of the protein and the DNA element is clearly asymmetric and hence distinct from that observed for the other two proteins. The fact that GBF1, GBF2 and GBF3 possess both distinct DNA binding properties and expression characteristics prompt us to entertain the notion that these proteins may individually mediate distinct subclasses of expression properties assigned to the G‐box. Furthermore, we demonstrate that GBF1, GBF2 and GBF3 heterodimerize and these heterodimers also interact with the G‐box, suggesting a potential mechanism for generating additional diversity from these GBF proteins.

Interleukin-4 (IL-4) stimulation leads to the activation of the signal transducer and activator of transcription 6 (Stat6). In this study, we present data relating to the functional properties of Stat6. Human embryonic kidney 293 cells were shown to be deficient of Stat6 yet express all other components of the IL-4 signaling cascade. This cell line was used for transient-transfection studies of wild-type and mutant Stat6 proteins. The wild-type protein was shown to activate a reporter construct carrying multiple copies of the IL-4 response element derived from the human immunoglobulin heavy-chain germ line epsilon promoter. Similarly, a truncated protein lacking 41 amino acids of the N terminus was fully active. However, removal of the C-terminal 186 amino acids completely abolished transcription activation. Amino acid substitutions were introduced into the putative DNA binding domain (VVI at positions 411 to 413), the SH2 domain (R-562), or the tyrosine (Y-641) which presumably becomes phosphorylated upon activation. All three of these Stat6 mutants were unable to activate transcription in 293 cells. Wild-type and mutant Stat6 derivatives were also expressed in insect cells, and purified proteins were analyzed in vitro for the ability to interact with both DNA and tyrosine-phosphorylated peptides derived from the IL-4 receptor alpha chain. Mutations within the DNA binding domain, the SH2 domain, or tyrosine 641 completely abolished DNA binding. In contrast, only the SH2 mutant failed to interact with tyrosine-phosphorylated peptides. The transdominant effects of all Stat6 derivatives were analyzed by using HepG2 cells, which express endogenous Stat6 protein. Differential effects were observed with various mutants, supporting the current model of the Jak/STAT activation cycle.

Transcription of the gene encoding the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) is induced in response to various cytokines, including tumor necrosis factor-et (TNF-a) and interleukin-1. A DNase I-hypersensitive site in the 5' proximal promoter region of the E-selectin gene is observed in human umbilical vein endothelial cells only following TNF-a treatment, suggesting the presence of a TNF-ot-inducible element close to the transcriptional start site. Transient transfection studies in endothelial cells demonstrated that 170 bp of upstream sequences is suflicient to confer TNF-a inducibility. Systematic site-directed mutagenesis of this region revealed two regulatory elements (-129 to -110 and -99 to -80) that are essential for maximal promoter activity following cytokine treatment. Protein binding studies with crude nuclear extracts and recombinant proteins revealed that the two elements correspond to three NF-KB binding sites (site 1, -126; site 2, -116; and site 3, -94). All three sites can be bound by NF-KB when used as independent oligonucleotides in mobility shift assays. However, within the context of a larger promoter fragment, sites 2 and 3 are preferentially occupied over site 1. These data are consistent with results obtained in transfection studies demonstrating that mutations in sites 2 and 3 are more detrimental than mutations within site 1. Hence, inducibility of the E-selectin gene requires the interaction of NF-KB proteins bound to multiple regulatory elements.

Latent and activated forms of Stat1 and Stat6 have been expressed and purified, enabling biochemical experiments relating to their functional activities. Stat1 bound to a phosphotyrosine peptide derived from the IFN gamma receptor with a KD of 50 nM, whereas Stat6 bound to an IL-4 receptor peptide with a KD of 300 nM. Stat-receptor peptide interactions were specific and dependent upon tyrosine phosphorylation. Activated forms of Stat1 and Stat6 were used to select their optimal DNA binding sites. Stat1 selected a recognition site having dyad half-sites separated by 3 bp. Stat6 selected a recognition site composed of the same dyad half-sites, yet separated by 4 bp. Chimeric Stat1-Stat6 recombinants were expressed, purified, and assayed for receptor coupling and DNA binding specificity. Such studies led to the identification of polypeptide domains that specify these activities. These observations provide a framework for understanding how different cytokines elicit distinctive patterns of gene expression.

Homeodomain proteins have been shown to play a major role in the development of various organisms. A novel Arabidopsis homeodomain protein has been isolated based on its capability to interact with a DNA motif derived from the light-induced cab-E promoter of Nicotiana plumbaginifolia. The homeodomain of this protein, designated HAT3.1, differs substantially from those in other plant homeobox proteins identified so far. Furthermore, HAT3.1 is unique among other Arabidopsis proteins in that it does not contain a leucine zipper motif following the homeodomain. HAT3.1 is further characterized by an N-terminal region that shares substantial sequence similarity with the maize homeodomain protein Zmhox1a. Within this conserved region, the presence of eight regularly spaced cysteine/histidine residues was observed reminiscent of other metal-binding domains. Based on the strong evolutionary conservation of this domain, it is proposed that this region represents a novel protein-motif which is denoted PHD-finger (plant homeodomain-finger). In vitro DNA binding studies demonstrated that HAT3.1 is capable of interacting with any DNA fragment larger than 100 bp. Interestingly, a deletion of the N-terminal PHD-finger domain completely abolished DNA binding, suggesting that this region may play an important functional role in protein-protein or protein-DNA interaction. HAT3.1 mRNA was primarily detected in root tissue, implying a regulatory function of this protein in root development.