Sp3 Is a Bifunctional Transcription Regulator with Modular Independent Activation and Repression Domains
Journal of Biological Chemistry
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
... 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 DNA-binding 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-terminal region. The negative regulatory function has been mapped 5Ј of the zinc finger region. Moreover, we demonstrated that the Sp3-repression ability is strikingly dependent upon the context of the Sp3 DNA-binding sites present in the reporter promoters. Sp3 functions as a repressor when it is bound to the promoter through multiple DNA-binding sites. Conversely, Sp3 turns out to be an activator when it is targeted to the promoter via a single DNA-binding site. In addition, using the Drosophila Schneider SL2 cells that lack endogenous Sp activity, we demonstrate that Sp3 does not possess an active repression domain that can function in insect cells, but rather it is a weak transcriptional activator of the human c-myc promoter, and it functionally co-operates in vivo with the TATAbinding general transcription factor TBP. 1 Our results suggest that Sp3 is a bifunctional transcriptional regulator and its predominant effect would depend on the context of Sp3 DNA-binding sites and on the nature of a corepressor present in a particular cellular background. EXPERIMENTAL PROCEDURES Reporter Plasmids-The Ϫ83HIV CAT reporter has been previously described (8), the GC-38HIV CAT is a derivative of the G5-38HIV (8) in which an oligonucleotide containing a single GC box was inserted into the XbaI site at Ϫ38 of the HIV promoter sequences. The pmyc-X⌬N is a reporter plasmid containing the human c-myc promoter sequences from the XhoI-NaeI fragment spanning from Ϫ93 to ϩ54 of the P2 transcription start site, and the pmyc-X⌬Nmut is the isogenic plasmid in which the single Sp1/Sp3-binding site located at position Ϫ53 was mutated by changing the CT box in a sequences containing adjacent SphI/XbaI sites as described previously (9). The G5-X⌬N and G1-X⌬N contain five or a single GAL4-binding site inserted at the XhoI site of the pmyc-X⌬N reporter, respectively. The X⌬N-G5 and X⌬N-G1 reporters contain five or a single GAL4-binding site inserted in the SphI/XbaI sites present in the pmyc-X⌬Nmut. The 3xCT-X⌬N has been constructed by inserting in the SphI/XbaI sites present in the pmyc-X⌬Nmut, an oligonucleotide containing a triplicated CT box. The G5E1b (11) and G1E1b are CAT reporter plasmids containing five or one GAL4-binding site upstream from the E1b TATA box, respectively.