NF-B induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-B-responsive genes are activated rapidly after the cell is stimulated by cytokines and other extracellular signals. However, the mechanism by which these genes are activated is not entirely understood. Here we report that even though NF-B interacts directly with TAF II s, induction of NF-B by tumor necrosis factor alpha (TNF-␣) does not enhance TFIID recruitment and preinitiation complex formation on some NF-B-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-␣ stimulus. Using the immediate-early TNF-␣-responsive gene A20 as a prototype promoter, we found that the constitutive association of the general transcription apparatus is mediated by Sp1 and that this is crucial for rapid transcriptional induction by NF-B. In vitro transcription assays confirmed that NF-B plays a postinitiation role since it enhances the transcription reinitiation rate whereas Sp1 is required for the initiation step. Thus, the consecutive effects of Sp1 and NF-B on the transcription process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.The family of NF-B transcription factors is a central component of the cellular response to a broad range of extracellular signals, many of them are related to immunological functions and stress. NF-B controls the expression of a large number of genes including inflammatory cytokines, chemokines, immunological factors, adhesion molecules, cell cycle regulators, and pro-and antiapoptotic factors (24). A major pathway regulating NF-B activity involves its nuclear transport. In unstimulated cells, NF-B is retained in the cytoplasm in an inactive form by IB proteins. Signals that activate NF-B trigger ubiquitination and degradation of IB by the proteosome, resulting in transport of NF-B into the nucleus and transcriptional activation of responsive genes. Since IB␣ is one of the NF-B target genes, the newly synthesized IB␣ negatively regulates NF-B, thus forming an autoregulatory loop.In the nucleus, transcriptional activation by NF-B involves its association with multiple coactivators. We reported previously that the substoichiometric TFIID subunit, TAF II 105, which is enriched in B cells, interacts directly with p65/Re1A, a member of the NF-B family, and is important for activation of a subset of NF-B-dependent antiapoptotic genes in vivo (30,36,37). Likewise, other TFIID subunits such as hTAF II 250, hTAF II 80, and hTAF II 28 were reported to bind to p65/Re1A (8), although the physiological importance of these interactions was not investigated. In addition to TFIID, the coactivator protein CREB-binding protein CBP and its homolog p300 were reported to be involved in transcription activation by the p65/Re1A subunit of NF-B (6, 25). p65 was also found to interact specifically with the composite coactivator ARC/DRIP, and this complex supports NF-B-dependent transcriptional activation in v...

A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNAcontacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.Transcription of protein-encoding genes in eukaryotes involves the assembly of RNA polymerase II and general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). TFIID is the major DNA-binding GTF. It is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TAFs regulate transcription at multiple levels. Certain TAFs interact with activators to facilitate PIC formation (2, 18) and transcription reinitiation (1). TAFs also have a role in recognition and binding to core promoter elements. DNase I footprinting revealed direct contact of TAFs with sequences upstream and downstream of the TATA box (11,19,23,26,29,35). TAF1-TAF2 (TAF II 250-TAF II 150) binds the Initiator element (7,33,34), multiple TAFs were cross-linked to the adenovirus major late (AdML) promoter (24), and Drosophila melanogaster TAF6 (dTAF6) and dTAF9 (dTAF II 60-dTAF II 42) were cross-linked to the downstream promoter element (DPE) (5). Some TAFs are TFIID specific, but others are shared by other transcription regulatory complexes (3,4,15,22,25,28).One common feature found in 9 out of the 14 TAFs is the histone fold motif (for a review see reference 13). This motif has been established as an essential protein-protein interaction domain that facilitates assembly of TFIID in a manner analogous to that for histones (17,30). TAF6 and TAF9 are structurally related to histones H4 and H3, respectively (38). TAF12 is similar to H2B, TAF4 contains an H2A-like domain, and both interact with each other via the histone fold domain (HFD) (14,16,30). In vitro Saccharomyces cerevisiae TAF6 (yTAF6)-yTAF9 can assemble with yTAF12-yTAF4 to form a histone octamer-like structure (30).The nucleosome-like interaction of TFIID with DNA (24) and the presence of histone fold TAFs within this complex have led to the proposal that a nucleosome-like octamer within TFIID may be involved in direct DNA binding (17). However, the issue has remained elusive. First, there is no experimental evidence for D...

Xenopus oocytes respond to trypsin with a characteristic chloride current, virtually indistinguishable from responses mediated by a large number of native and expressed G protein-coupled receptors. We studied the involvement of G proteins of the G␣ q family as possible mediators of this and other G protein-coupled receptormediated responses in Xenopus oocytes. We have cloned the third member of the G␣ q family, Xenopus G␣ 14 , in addition to the previously cloned Xenopus G␣ q and G␣ 11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89 -92). Amphibian G␣ 14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the G␣ 14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiG␣ 14 ) that, together with antiG␣ q and antiG␣ 11 , was used in antisense depletion experiments. 24 h after injection into oocytes, either antiG␣ q or antiG␣ 14 reduced the response to 1 g/ml trypsin by 70%, whereas antiG␣ 11 had no effect. A mixture of antiG␣ q and antiG␣ 14 virtually abolished the response. These data strongly suggest that G␣ q and G␣ 14 are the exclusive mediators of the trypsin-evoked response in Xenopus oocytes. Similar experiments with the expressed gastrin-releasing peptide receptor and muscarinic m1 receptor revealed the coupling of G␣ q and G␣ 11 , but not G␣ 14 , to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the G␣q family discriminate among different native receptors in vivo.Heterotrimeric GTP-binding proteins (G proteins) of the G␣ q family activate the phosphatidylinositol-bisphosphate-inositoltrisphosphate-calcium pathway in a pertussis toxin-insensitive manner. Among G␣ q family members, G␣ q and G␣ 11 share 88% homology and are expressed ubiquitously (2-4). G␣ 14 is 81% identical to G␣ q and is restricted in its tissue distribution mainly to spleen, lung, kidney, and testes (5). G␣ 15 and its human counterpart, G␣ 16 , show only 58 and 57% amino acid identity, respectively, to mouse G␣ q and are restricted to a subset of hematopoietic cells (5, 6). Upon activation, all G proteins of the G␣ q family activate ␤ isomers of phospholipase C.The coexistence of closely related G proteins in the same cell suggests different functions or different interactions between individual G proteins and either receptors or phospholipases. However, in reconstitution systems and transfected cells, G␣ q and G␣ 11 have been shown to couple indiscriminately to a wide range of receptors, e.g. muscarinic m1 receptor (m1-R) 1 (7, 8) and m3-R (9), thyrotropin-releasing hormone (10, 11), parathyroid hormone (12), gastrin-releasing peptide (GRP) and vasopressin (13), gonadotrophin-releasing hormone (14), angiotensin II and bradykinin (15), histamine (16), and ␣ 1 -adrenergic receptors (17). G␣ 15/16 , despite restricted distribution, are capable of coupling many serpentine receptors tested, including those natively coupled to G␣ s and G␣ i (for review, see Ref. 18). Similarly, an attempt to assign differ...

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