FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes, possesses potent transcription activation and repression domains, and is necessary for c-myc expression. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocked activator-dependent, but not basal, transcription through TFIIH. Recruited through FBP's nucleic acid-binding domain, FIR formed a ternary complex with FBP and FUSE. FIR repressed a c-myc reporter via the FUSE. The amino terminus of FIR contained an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targeted only TFIIH's p89/XPB helicase, required at several stages in transcription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription.

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c-myc and p53 networks control proliferation, differentiation, and apoptosis and are responsive to, and cross-regulate a variety of stresses and metabolic and biosynthetic processes. At c-myc, the far upstream element binding protein (FBP) and FBP-interacting repressor (FIR) program transcription by looping to RNA polymerase II complexes engaged at the promoter. Another FBP partner, JTV1/AIMP2, a structural subunit of a multi-aminoacyl-tRNA synthetase (ARS) complex, has also been reported to stabilize p53 via an apparently independent mechanism. Here, we show that in response to oxidative stress, JTV1 dissociates from the ARS complex, translocates to the nucleus, associates with FBP and co-activates the transcription of a new FBP target, ubiquitin-specific peptidase 29 (USP29). A previously uncharacterized deubiquitinating enzyme, USP29 binds to, cleaves poly-ubiquitin chains from, and stabilizes p53. The accumulated p53 quickly induces apoptosis. Thus, FBP and JTV1 help to coordinate the molecular and cellular response to oxidative stress.

A family of tumor necrosis factor receptor-associated factors (TRAFs 1-7) 1 functions as adaptor molecules for TNF receptor superfamily members by associating with the intracellular domain of these proteins and subsequently mediating downstream signaling events such as NF-B and AP-1 (1, 2). Biochemical approaches have revealed that TRAFs form homotypic multimers (3-6) as well as certain heterotypic multimers, such as those between TRAF1 and TRAF2 and between TRAF3 and TRAF5 (7-10).Previous reports have demonstrated that TRAF2 and -3 play an important role in cellular activation and differentiation following engagement of a variety of TNF receptor superfamily members such as CD40/TNFRSF5 (1), OX40/TNFRSF4 (11), LT␤R (12-14), XEDAR (15), BCMA/TNFRSF17 (16), and Fn14/ TWEAKR/TNFRSF12A (17, 18). The functional significance of TRAF2 and -3 in immune responses mediated by one or more of these TNF receptor superfamily molecules was revealed by experiments with mice that were genetically altered in expression of TRAF2 or -3. Experiments using mice transgenic for only the TRAF domain of TRAF2 (amino acids 245-501; TRAF2.dominant negative; Ref. 19) or mice genetically deficient in TRAF3 expression (20) revealed a role for both adaptor proteins in T cell-dependent humoral immune responses. Of note, mice expressing a dominant negative form of TRAF2 exhibited an expanded B cell compartment that was evidenced by splenomegaly and lymphadenopathy (21), whereas TRAF3Ϫ/Ϫ mice exhibited decreased numbers of B cell precursors (20). Examination of signaling mechanisms mediated by TRAF2 or -3 revealed that both adaptor proteins induce activation of the mitogen-activated protein kinase Jun N-terminal kinase (JNK) as well as playing a role in the regulation of . Importantly, a number of reports have indicated that TRAF3 inhibits NF-B activation induced by TRAF2 following engagement of TNF receptor superfamily members such as CD40/TNFRSF5 (29) and OX40/TNFRSF4 (30,31), but the precise mechanism of this observation has not been delineated. However, overexpression of wild-type TRAF3 has been shown to inhibit TRAF2-induced 31). Furthermore, proteolysis of TRAF3 by a pepstatin A inhibitable mechanism enhanced CD40-mediated NF-B activation (32), whereas TRAF2-induced degradation of TRAF3 enhanced . By contrast, expression of an alternatively spliced form of TRAF3 has been shown to activate 35). These findings suggest that a complex role for TRAF2 and TRAF3 in the regulation of NF-B, but the precise molecular mechanism has not been delineated. Importantly, no direct physical interaction between TRAF2 and TRAF3 has been documented to date.The purpose of the current study was to examine whether functional inhibition of TRAF2-induced NF-B activation was mediated by a direct interaction between TRAF2 and -3. Experiments utilizing one-and two-step FRET performed by confocal microscopy or flow cytometry clearly demonstrated that TRAF3 directly associates with TRAF2 and inhibits TRAF2-induced NF-B, but not AP-1, activation. MATERIALS AND METHODSPlas...