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Enhancers when functioning at a distance cannot effectively stimulate transcription from core promoters. We demonstrate that this is due to the inability of enhancer-bound activators to recruit TBP to a distal TATA box. Surprisingly, binding of a transcriptionally inert Oct-1 POU domain near a core promoter enables an enhancer to function from a distance. POU activity neither requires the coactivator OCA-B nor the interaction of TBP with TFIIA. Instead, the POU domain directly facilitates TBP recruitment to the promoter utilizing a bipartite interaction surface. These results establish that an interaction between the DNA binding domain of an activator and TBP can be used to stimulate transcription. Furthermore, they suggest a mechanism for long-range enhancer function in which a TBP complex is preassembled on a promoter via localized recruitment and then acted upon by distal activators.

The X-ray structure of a ternary complex of Negative Cofactor 2 (NC2), the TATA box binding protein (TBP), and DNA has been determined at 2.6 A resolution. The N termini of NC2 alpha and beta resemble histones H2A and H2B, respectively, and form a heterodimer that binds to the bent DNA double helix on the underside of the preformed TBP-DNA complex via electrostatic interactions. NC2beta contributes to inhibition of TATA-dependent transcription through interactions of its C-terminal alpha helix with a conserved hydrophobic feature on the upper surface of TBP, which in turn positions the penultimate alpha helix of NC2beta to block recognition of the TBP-DNA complex by transcription factor IIB. Further regulatory implications of the NC2 heterodimer structure are discussed.

Transcription of TATA box-containing genes by RNA polymerase II is mediated by TBP-containing and TBP-free multisubunit complexes consisting of common and unique components. We have identified a highly stable TBP-TFIIA-containing complex, TAC, which is detectable in embryonal carcinoma (EC) cells but not in differentiated cells. TAC contains the TFIIAgamma subunit and the unprocessed form of TFIIAalphabeta, although the processed TFIIAalpha and TFIIAbeta subunits are present in EC cells. TAC mediates transcriptional activation by RNA polymerase II in vivo, even though it does not contain classical TAFs. Formaldehyde cross-linking revealed that in EC but not in differentiated cells, association of TBP with chromatin is strongly enhanced when complexed with TFIIA in vivo. Remarkably, the TFIIAalphabeta precursor is preferentially, if not exclusively, associated with chromatin as compared to the processed subunits present in "free" TFIIA in EC cells.

Dimerization of the TATA-binding protein (TBP) through its DNA-binding domain blocks TBP from accessing DNA and prevents unregulated gene expression. TFIIA plays a central role in loading TBP and its multisubunit counterpart TFIID onto promoter DNA, and it is therefore a candidate for regulating TBP/TFIID dimerization. Here, we show that TFIIA promotes the dissociation of TBP dimers directly and in doing so accelerates the kinetics of DNA binding. TFIID dimer dissociation was found to be slow and rate limiting in DNA binding. TFIIA induced a rapid dissociation of TFIID dimers, allowing TFIID to readily load onto promoter DNA. Together, these results suggest a novel mechanism by which TFIIA assists in regulating gene expression.

The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor, critical for normal cell cycle progression. We have undertaken studies using a highly purified reconstituted in vitro transcription system to demonstrate how pRB can repress transcriptional activation mediated by the E2F transcription factor. Remarkably, E2F activation became resistant to pRB-mediated repression after the establishment of a partial (TFIIA/TFIID) preinitiation complex (PIC). DNase I footprinting studies suggest that E2F recruits TFIID to the promoter in a step that also requires TFIIA and confirm that recruitment of the PIC by E2F is blocked by pRB. These studies suggest a detailed mechanism by which E2F activates and pRB represses transcription without the requirement of histone-modifying enzymes.

A functional interferon-beta gene enhanceosome was assembled in vitro using the purified recombinant transcriptional activator proteins ATF2/c-JUN, IRF1, and p50/p65 of NF-kappa B. Maximal levels of transcriptional synergy between these activators required the specific interactions with the architectural protein HMG I(Y) and the correct helical phasing of the binding sites of these proteins on the DNA helix. Analyses of the in vitro assembled enhanceosome revealed that the transcriptional synergy is due, at least in part, to the cooperative assembly and stability of the complex. Reconstitution experiments showed that the formation of a stable enhanceosome-dependent preinitiation complex require cooperative interactions between the enhanceosome; the general transcription factors TFID, TFIIA, and TFIIB; and the cofactor USA. These studies provide a direct biochemical demonstration of the importance of the structure and function of natural multicomponent transcriptional enhancer complexes in gene regulation.

Eukaryotic cells are thought to contain a single TATA-binding protein (TBP) that directs transcription by cellular RNA polymerases. Here we report a cell type-specific TBP-related factor (TRF) that can form a stable TRF/IIA/IIB TATA DNA complex and substitute for TBP in directing RNA polymerase II transcription in vitro. Transfection studies reveal that TRF can differentially mediate activation by some enhancer proteins but not others. Like TBP, TRF forms a stable complex containing multiple novel subunits, nTAFs. Antibody staining of embryos and polytene chromosomes reveals cell type-specific expression and gene-selective properties consistent with the shaker/male sterile phenotype of trf mutants. These findings suggest TRF is a homolog of TBP that functions to direct tissue- and gene-specific transcription.

Four new studies of the assembly of different subsets of the preinitiation complex of RNA polymerase II have provided insight into how transcription factors interact with each other and with DNA. A heterotetramer of TBP-associated factors shows a histone-like fold.

The D. melanogaster alcohol dehydrogenase (Adh) gene is transcribed from two tandem promoters that are differentially utilized at various stages during development. To determine the mechanism of promoter selectivity, we have analyzed the activity of the Adh promoters both in vitro and in transfected cells. We found that selective promoter utilization is controlled by distinct initiator elements. Reconstitution of Adh transcription with purified components requires a specific TBP-TAF complex that, in concert with TFIIA, directs differential Adh promoter transcription. Fractionation of this TBP-TAF complex reveals that TAFII150 is required for discrimination between the proximal and distal promoters. We propose a mechanism for regulating differential promoter utilization during Drosophila development that involves the recognition of specific initiator elements by TAFs in the TFIID complex.

Activator-dependent transcription in mammalian cells requires upstream stimulatory activity (USA)-derived cofactors in addition to those present in TFIID. A novel positive cofactor (PC4) purified from the human USA fraction effected a marked enhancement (up to 85-fold) of GAL4-AH-dependent transcription in conjunction with TFIID and other general factors. Isolation of a corresponding cDNA identified PC4 as a 127 residue single-stranded DNA-binding protein with serine-rich regions near the N-terminus. Recombinant PC4 was functionally equivalent to native PC4, and both proteins markedly enhanced activation by diverse activation domains fused to the DNA-binding domain of GAL4. Recombinant PC4 interacted independently both with free or DNA-bound VP16 activation domains and with free or DNA-bound TFIIA-TBP complexes (but not with TBP alone). These results indicate that PC4 is a general coactivator that functions cooperatively with TAFs and mediates functional interactions between upstream activators and the general transcriptional machinery.

Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled. Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ. Promoters vary in their activities in the minimal reaction. Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density. When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription. It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors.

Acetylation of the N-terminal tails of the core histones directly facilitates the recognition by TFIIIA of the 5S RNA gene within model chromatin templates. This effect is independent of a reduction in the extent of histone-DNA interactions or a change in DNA helical repeat; it is also independent of whether a histone tetramer or octamer inhibits TFIIIA binding. Removal of the N-terminal tails from the core histones also facilitates the association of TFIIIA with nucleosomal templates. We suggest that the histone tails have a major role in restricting transcription factor access to DNA and that their acetylation releases this restriction by directing dissociation of the tails from DNA and/or inducing a change in DNA configuration on the histone core to allow transcription factor binding. Acetylation of core histones might be expected to exert a major influence on the accessibility of chromatin to regulatory molecules.

We have discovered a protein termed Dr1 that interacts with the TATA-binding protein, TBP. The association of Dr1 with TBP results in repression of both basal and activated levels of transcription. The interaction of Dr1 with TBP precludes the formation of a transcription-competent complex by inhibiting the association of TFIIA and/or TFIIB with TBP. Dr1 activity is associated with a 19 kd protein. A cDNA clone encoding Dr1 was isolated. Dr1 is phosphorylated in vivo and phosphorylation of Dr1 affected its interaction with TBP. Our results suggest a regulatory role for Dr1 in repression of transcription mediated via phosphorylation.

A family of proteins was shown to bind cooperatively with TFIID to core promoters, as previously demonstrated for the general initiation factor TFIIA. These factors form distinct complexes with TFIID, fail to bind DNA in the absence of TFIID, differ chromatographically from TFIIA, and compete with TFIIA for binding to TFIID. Our results suggest the formation of heterogeneous preinitiation complexes at the step involving TFIIA interactions. This establishes a molecular switch that regulates basal level transcription in vitro and has consequences for transcriptional activation by gene-specific activators.