These results indicate that Vav is required for cap formation in lymphocytes. Furthermore, the correlation between cap formation, IL-2 production and proliferation supports the hypothesis that an actin-dependent pathway is a source of specialized growth regulatory signals.

One of the important regulatory concepts to emerge from studies of eukaryotic gene expression is that RNA polymerase II promoters and their upstream activators are composed of functional modules whose synergistic action regulates the transcriptional activity of a nearby gene. Biochemical analysis of synergy by ZEBRA, a non-acidic activator of the Epstein-Barr virus (EBV) lytic cycle, showed that the synergistic transcriptional effect of promoter sites and activation modules correlates with assembly of the TFIID:TFIIA (DA) complex in DNase I footprinting and gel shift assays. The activator-dependent DA complex differs from a basal DA complex by its ability to bind TFIIB stably in an interaction regulated by TATA-binding protein-associated factors (TAFs). TFIIB enhances the degree of synergism by increasing complex stability. Similar findings were made with the acidic activator GAL4-VP16. Our data suggest a unifying mechanism for gene activation and synergy by acidic and non-acidic activators, and indicate that synergy is manifested at the earliest stage of preinitiation complex assembly.

The SWI͞SNF complex in yeast and Drosophila is thought to facilitate transcriptional activation of specific genes by antagonizing chromatin-mediated transcriptional repression. The mechanism by which it is targeted to specific genes is poorly understood and may involve direct DNA binding and͞or interactions with specific or general transcription factors. We have previously purified a mammalian complex by using antibodies against BRG1, a human homologue of SWI2͞SNF2. This complex is likely functionally related to the yeast SWI͞SNF complex because all five subunits identified so far (referred to as BAFs, for BRG1-associated factors) are homologues of the yeast SWI͞SNF subunits. However, we now describe the cloning of the 57-kDa subunit (BAF57), which is present only in higher eukaryotes but not in yeast. BAF57 is shared by all mammalian complexes and contains a high-mobility-group (HMG) domain adjacent to a kinesin-like region. Both recombinant BAF57 and the whole complex bind four-way junction (4WJ) DNA, which is thought to mimic the topology of DNA as it enters or exits the nucleosome. Surprisingly, complexes with mutations in the HMG domain of BAF57 can still bind 4WJ DNA and mediate ATP-dependent nucleosome disruption. Our work describes the first DNA binding subunit for SWI͞SNF-like complexes and suggest that the mechanism by which mammalian and Drosophila SWI͞SNF-like complexes interact with chromatin may involve recognition of higher-order chromatin structure by two or more DNA binding domains.

The prevailing view of eukaryotic gene activation poses that activators stimulate transcription by recruiting limiting components of the general transcription machinery to a core promoter. In one such model case, activation by the Epstein-Barr virus ZEBRA protein correlated closely with recruitment of the general transcription factors TFIIA and TFIID (the DA complex) as measured by DNase I footprinting and gel mobility shift assays. We now report that simple recruitment is not sufficient for full-level activation. An additional concentration-independent, rate-limiting step is activator-mediated isomerization of the DA complex characterized by an extended TFIID footprint. The isomerized complex supports both binding of TFIIB in gel mobility shift assays and activated transcription in heat-treated nuclear extracts, even after removal of ZEBRA. Surprisingly, the regulatory phenomenon of synergy was manifested only when the concentration of TFIID was limiting. When the DA complex was saturating, transcription was not synergistic, as indicated by the ability of a single activator to induce isomerization effectively and turn on a gene. On the basis of these observations, we propose a new biochemical model for eukaryotic gene activation and synergy. The question of how activators stimulate transcription has dominated the field of eukaryotic gene regulation throughout the last decade. The complexity of the problem is emphasized by the paradoxically simple organization of upstream activator proteins and the large size and complexity of the RNA polymerase II (pol II)transcription machinery. The details of how activators contact the transcription machinery and the consequences of such interactions have provided a multidisciplinary challenge encompassing both biochemistry and genetics.

The TISll primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The TIS11 5' primer. We conclude that the original TIS11 cDNA is likely to be the product of a cloning artifact that created a chimeric cDNA molecule. The PCR product was cloned into M13 and sequenced. The additional C residue reported in TTP (13) was clearly present and shifted the TIS11 reading frame to give a protein whose C-terminal sequence is identical to that of TTP/Nup475 (5, 13). Our sequence of the 5' region of the cloned cDNA from the PCR was identical to that described for TTP/Nup475 (Fig. 1). Antiserum to recombinant Nup475 protein detects transiently elevated nuclear staining in serum-treated cells (5). The repeated Cys-His motif, serine-proline-rich domains, and nuclear localization all suggest that the TIS11/TTPINup475 protein may be a rapidly inducible transcription factor.Cloning and sequence of TISllb cDNA. Arguing by analogy to the importance of conserved amino acid sequence motifs within the fos, jun, and egr families of primary response genes (8)

An RNA polymerase II activator often contains several regions that contribute to its potency, an organization ostensibly analogous to the modular architecture of promoters and enhancers. The regulatory significance of this parallel organization has not been systematically explored. We considered this problem by examining the activation domain of the Epstein-Barr virus transactivator ZEBRA. We The ATP requirement may be for TFIIH, which acts as both a kinase and an ATP-dependent helicase (31).Activators are often classified according to the amino acid composition of their activation domains or regions. It has been proposed that different classes of activation domains may function by different mechanisms (37). Acidic activation regions are rich in acidic amino acids and have a net negative charge. Recent mechanistic studies have shown that GAL4-derived acidic activators (5, 28), containing the GAL4 DNA binding domain fused to various acidic activation regions (41), act by promoting transcription complex assembly (29,54). Furthermore, the synergistic effect of the GAL4 derivatives on transcription (a single activator is either impotent or marginally active, whereas two or more activators elicit a greater than additive response [5]) correlates with a synergistic increase in the number of open complexes (55). Recruitment and kinetic experiments suggest that acidic activators target early steps in preinitiation complex formation (8,18,29,47,53,55,56), including assembly of subcomplexes requiring TFIIA and TFIID (55) or TFIIB and TFIID (29,47). Although a mechanism for how acidic activators function is emerging, little is known about how other classes of activation domains work. It is noteworthy, however, that affinity chromatography and far-Western blotting experiments have implicated factors involved in early steps in complex assembly as potential targets of both acidic and nonacidic activators (23,25,27,30,46).An activator often contains two or more activation regions, or subdomains, distinguished by their locations within the activator and by their chemical compositions (37). Depending upon the particular study, these activation regions act either synergistically, additively, or redundantly to influence the activator's potency (13,17,21,32,38,48,49,52). It has been noted (13) that the organization and function of the multiple activation regions are superficially analogous to the modular structure of promoters and enhancers (7,39, 7045 on May 9, 2018 by guest

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