SP1 TRANSACTIVATION OF CELL-CYCLE-REGULATED PROMOTERS IS SELECTIVELY REPRESSED BY SP3

The transcription factor Spl plays a key role in the activation of man y cellular and viral gene promoters, including those that are regulate d during the cell cycle. However, recent evidence indicates that Sp be longs to a larger family of factors which bind G/C box elements in ord er to either activate or repress transcription. Sp3, a member of this family, functions to repress transcriptional activation in two viral p romoters, most likely by competing with Spl for GC box/Sp binding site s. However, the physiological role of Sp3 in the repression of endogen ous cellular promoters has not been experimentally addressed. In the p resent study, we analyze the activity and binding of Sp3 on several eu karyotic promoters that contain G/C bares and are known to be regulate d during cellular proliferation and the cell cycle. Using antibodies s pecific for Spl and Sp3, we observe that both of these factors localiz e to the cell nucleus and have a similar, dispersed subnuclear distrib ution. Further, using gel mobility shift assays, we show that both Spl and Sp3 interact specifically with the histone H4 promoter. Transient cotransfections of Drosophila cells with Spl and Sp3 expression vecto rs and with the histone H4, thymidine kinase (TK), or dihydrofolate re ductase (DHFR) promoters show that only the DHFR promoter, containing multiple functional GC boxes, displays Sp3 repression of Spl activatio n. In contrast, the single G/C boxes within the histone H4 or TK promo ters, which confer transcriptional activation via Spl binding, are not responsive to repression by Sp3. Therefore, we demonstrate that the e ndogenous cellular DHFR promoter is selectively responsive to Sp3 repr ession. The data suggest that Sp3 may contribute to the control of pro liferation- and/or cell cycle-regulated promoters depending upon the c ontext and/or number of functional Spl binding sites.