No abstract

Recently, a family of transcription factors structurally related to Sp1 has been described; thus, more than one activator may bind to the GC boxes present in a number of viral and cellular promoters. We have compared the transactivation potentials of Sp1, Sp3 and Sp4 proteins on the human immunodeficiency virus type 1 (HIV-1) promoter. The long terminal repeat (LTR) of HIV-1 contains three binding sites for the transcription factor Sp1 (GC boxes) which are involved in both basal and Tat-mediated transcriptional activation. Moreover, a cooperative interaction between NF-kappa B and Sp1 is required for HIV enhancer activation. We now demonstrate that Sp4 is an activator, while the Sp3 protein represses basal expression of HIV promoter. Remarkably, we found that over-expression of the transcription factor Sp3 was able to suppress Tat-mediated transactivation. These inhibitory effects of Sp3 correlate with its DNA binding activity, suggesting that Sp3 inhibition involves competition with Sp1 for occupancy of the GC boxes. Next, we have analyzed the role of different Sp1-related proteins in the stimulation of HIV-1 promoter in response to mitogens. We found that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, an interaction between NF-kappa B and the trans-acting domain (A domain) of Sp1 bound to an adjacent site must occur. We found that the cooperative interaction between NF-kappa B and Sp1 is highly specific, since neither Sp3 nor Sp4 is capable of cooperating with NF-kappa B.

Sp3 is a member of the Sp family of transcription factors and binds to DNA with affinity and specificity comparable to that of Sp1. We demonstrate that Sp3 is a bifunctional transcription factor that can both activate and repress transcription. Gene fusion experiments in mammalian cells demonstrate that the Sp3 activation potential is distributed over an extensive glutaminerich N-terminal region, whereas the repressor activity has been mapped in a 72-amino acid region located at the 5 of the zinc finger DNA-binding domain. We demonstrated that the repression activity is strictly dependent on the context of the DNA-binding sites bound by Sp3. We found that Sp3 represses transcription of promoters bearing multiple GAL4 DNA-binding sites, whereas it activates isogenic reporters containing a single GAL4-binding site. Transfection experiments in Drosophila cells that lack endogenous Sp activity demonstrated that Sp3 does not possess an active repression domain that can function in insect cells, rather it is a weak transcriptional activator of the c-myc promoter. Our results strongly suggest that Sp3 is a dual-function regulator whose activity is dependent upon both the promoter and the cellular context. Sp1 is a ubiquitously expressed transcription factor that plays a major role in the regulation of a large number of gene promoters, including constitutive and inducible genes (1-3). Recently the existence of an Sp family of transcription factors has been documented (4, 5). Sp1, Sp3, and Sp4 proteins have similar structural features with highly conserved DNA-binding domains and they recognize GC and CT boxes with closely similar specificity and affinity (5-7). Moreover, it has been shown that both Sp1 and Sp3 proteins are ubiquitously expressed in many mammalian cell lines, whereas Sp4 expression appears to be restricted to certain cell types (5-7). Transfection experiments into mammalian and Drosophila SL2 cells have shown that Sp4, like Sp1, is a transcriptional activator of Sp1 responsive promoters, whereas Sp3 represses Sp1-mediated transcription (5-9). Clearly the existence of proteins similar to Sp1 suggests that gene regulation by Sp1 is more complex than previously assumed. We previously showed that transfer of the nonfinger region of the Sp3 protein to a heterologous DNAbinding domain confers repressor function to the fusion protein, allowing it to repress transcription from reporter promoters containing multiple DNA-binding sites. Our previous data suggest that repression by chimeric Sp3 proteins occurs via protein-protein interaction with components of the basal transcription complex (10).In the present study we have embarked on a detailed analysis of the Sp3 function. To investigate the transcriptional properties of Sp3 in mammalian cells we have used gene fusion experiments to dissect the functional domains of Sp3. We found that Sp3 is a bifunctional protein containing independent modular repressor and activator domains. The activation potential of Sp3 is distributed over an extensive glutamine-rich N-term...

No abstract