Basal Transcription Complex Research Articles

Negative regulation of Sp1 trans-activation is correlated with the binding of cellular proteins to the amino terminus of the Sp1 trans-activation domain.

Sp1 is a well characterized and ubiquitously expressed transcription factor that regulates the constitutive and induced expression of a variety of mammalian genes. It is unclear whether Sp1 activity is regulated in vivo; the mechanism by which Sp1 interacts with the basal transcription complex has not been firmly established. We report the identification of a ubiquitously expressed and evolutionarily conserved nuclear protein, p74, that specifically binds Sp1 in vivo and in vitro. p74 interacts with several portions of the Sp1 trans-activation domain in vitro, and we correlate the binding of p74 to the amino-terminal serine/threonine-rich subdomain of Sp1 with the inhibition of Sp1-mediated transcription in vivo.

Transcriptional regulation of the dihydrofolate reductase/rep-3 locus.

This review summarizes many studies that have used the mouse, hamster, or human dihydrofolate reductase locus as a model system for the study of basal or regulated transcription. This locus encodes two genes, dhfr and rep-3, that are oriented in opposite directions. The dhfr promoter and the two rep-3 promoters are highly GC-rich and do not contain consensus TATA boxes. The cis-acting elements important in basal transcription of these non-TATA box promoters have been mapped, revealing that Sp1 plays a dominant role, and that sequence elements both upstream and downstream of the initiation site contribute to transcriptional activity. Future studies will be directed toward understanding the assembly of transcription complexes on these promoters. The expression of the dhfr gene following treatment of cells with many different stimuli has been analyzed, and in many, but not all, cases the response has been at the transcriptional level. In those systems where the regulatory cis-acting element has been mapped, the E2F sites flanking the transcription initiation site play a central role in mediating the response. Future studies will be directed at characterizing the regulatory protein E2F and understanding how it interacts with the basal transcription complex at the dhfr promoter. The rep-3 gene contains two promoters that are both growth responsive: one promoter is regulated by E2F, the regulator of the other promoter has not been identified. In summary, the in-depth characterization of the 1 kb containing the dhfr and rep-3 promoters has begun to provide a detailed understanding of growth-responsive transcription.

Hexamethylene bisacetamide activates the human immunodeficiency virus type 1 provirus by an NF- B-independent mechanism

Expression of the human immunodeficiency virus type 1 (HIV-1) provirus in T lymphocytic and monocytic cells can be induced by treatment with hexamethylene bisacetamide (HMBA). The induction occurs at the transcriptional level within 1 to 3 h after the addition of the drug, and is not associated with detectable changes in the binding of transcription factors to the enhancer, TATA box or other regulatory regions of the HIV-1 long terminal repeat (LTR). Using the 5' deletion mutants of HIV-1 LTR controlling the expression of the chloramphenicol acetyltransferase gene, we found that the deletion of the kappa B enhancer did not affect HIV-1 inducibility, whereas the deletion of the Sp1 binding sites abolished transcriptional activation. However, the presence of the HIV-1 LTR Sp1 binding sites in the context of the heterologous promoter did not induce responsiveness to HMBA. We conclude that HMBA increases transcription through the secondary modification of the basal transcription complex suggesting the existence of a regulatory pathway that circumvents the requirement for the induction of NF-kappa B or other DNA-specific binding proteins.

Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.

Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.

Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus early region 3 promoter regulation by E1A/E1B is independent of alterations in DNA binding and gene activation of CREB/ATF and AP1

Transcription of the adenovirus early region 3 promoter is strongly induced by the adenovirus E1A protein. Previous DNase I footprinting has indicated that four regions in this promoter serve as binding sites for HeLa nuclear proteins. These include binding sites for NF-1 (site IV), AP1 (site III), CREB/activating transcription factor (ATF) (site II), and TATA (site I). To determine the relative importance of these sites in both the in vivo and in vitro transcriptional regulation of the E3 promoter, oligonucleotide-directed mutagenesis of these sites was performed. Each of these constructs was assayed by transfection onto HeLa cells in the presence of either dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. Mutations of either the ATF- or AP1-binding sites but not the TATA- and NF1-binding sites resulted in severe decreases in both basal and E1A/E1B-induced transcriptional levels. These constructs were also assayed in in vitro transcription assays with cellular extracts prepared from dl434-infected or wild-type-adenovirus-infected HeLa cells. The wild-type E3 promoter was transcribed approximately 30 times more efficiently in extracts containing the E1A/E1B proteins compared with extracts lacking these proteins. Mutations of either the TATA element, the ATF site, or the AP1-binding site decreased both basal and E1A/E1B-induced transcriptional levels. Gel retardation analysis using these extracts indicated that the binding to ATF, AP1, or NF1 oligonucleotides was not altered in the presence of the E1A/E1B proteins compared with extracts lacking these proteins. Northern (RNA) blot analysis of c-jun and CREB RNA prepared from wild-type adenovirus and dl434-infected cells indicated that the levels of these RNAs were not altered by the E1A/E1B proteins. Immunoprecipitation of AP1 and CREB from both dl434- and wild-type-adenovirus-infected cells indicated that the amounts of these proteins were not significantly altered. These results suggest that E1A/E1B-induced activation of the E3 promoter does not involve activation of transcription factor genes nor a change in the DNA binding activity of important promoter-binding components. Our results are consistent with a model in which the E1A/E1B proteins either directly or indirectly alter the interactions of factors that bind to the basal E3 promoter transcription complex, thereby inducing transcription.

E1a Transactivation of the Human HSP70 Promoter Is Mediated through the Basal Transcriptional Complex

We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.

E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex.

We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.