The activation of cytokine genes in response to antigenic stimulation of T cells is mediated by NF-AT proteins. Previous studies have identified two NF-AT proteins, NF-ATp and NF-ATc, that are homologous within a 290 aa domain distantly related to the Rel domain. We have isolated two additional members of this gene family, NF-AT3 and NF-AT4, which encode proteins 65% identical to the other NF-AT proteins within the Rel domain. The four NF-AT genes are transcribed in different sets of tissues that included many sites of expression outside the immune system. The Rel homology domain is sufficient for DNA recognition and cooperative binding interactions with AP-1. Although other members of the Rel family bind DNA as dimers, NF-AT proteins are monomers in solution or bound to DNA. Transfection assays indicate that each of the four NF-AT proteins can activate the IL-2 promoter in T cells.

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.

Tumor necrosis factor (TNF) receptorassociated factor (TRAF) proteins associate with and transduce signals from TNF receptor 2, CD40, and presumably other members of the TNF receptor superfamily. TRAF2 is required for CD40-and TNF-mediated activation of the transcription factor NF-#cB. Here we describe the isolation and characterization of a novel TRAF-interacting protein, I-TRAF, that binds to the conserved TRAF-C domain of the three known TRAFs. Overexpression of I-TRAF inhibits TRAF2-mediated NF-KB activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator ofTRAF function that may act by maintaining TRAFs in a latent state.

Brain development is critically dependent on thyroid hormone (T(3)). Krüppel-like factor 9 (Klf9) is a T(3)-inducible gene in developing rat brain, and several lines of evidence support that KLF9 plays a key role in neuronal morphogenesis. Here we extend our findings to the mouse and demonstrate the presence of a functional T(3) response element (T(3)RE) in the 5' flanking region of the mouse Klf9 gene. Klf9 mRNA is strongly induced in the mouse hippocampus and cerebellum in a developmental stage- and T(3)-dependent manner. Computer analysis identified a near optimal direct repeat 4 (DR-4) T(3)RE 3.8 kb upstream of the Klf9 transcription start site, and EMSAs showed that T(3) receptor (TR)-retinoid X receptor heterodimers bound to the T(3)RE with high affinity. The T(3)RE acts as a strong positive response element in transfection assays using a minimal heterologous promoter. In the mouse neuroblastoma cell line N2a[TRbeta1], T(3) caused a dose-dependent up-regulation of Klf9 mRNA. Chromatin immunoprecipitation assays conducted with N2a[TRbeta1] cells showed that TRs associated with the Klf9 T(3)RE, and this association was promoted by T(3). Treatment of N2a[TRbeta1] cells with T(3) led to hyperacetylation of histones 3 and 4 at the T(3)RE site. Furthermore, TRs associated with the DR-4 T(3)RE in postnatal d 4 mouse brain, and histone 4 acetylation was greater at this site compared with other regions of the Klf9 gene. Our study identifies a functional DR-4 T(3)RE located in the mouse Klf9 gene to explain its regulation by T(3) during mammalian brain development.

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