We have previously shown that mutations in the GGAA core motif of the Ets1 
binding site, EBSI, or deletion of EBSI, reduced basal and Tax1 transactivation 
of the PTHrP P2 promoter. Here we demonstrate that, in addition to EBSI, a 
CACCC-like motif located between -53 and -58 is required for full basal activity 
of this promoter in Jurkat T-cells. Site-specific mutations in the CACCC motif 
decreased promoter activity approximately 5-fold. In an effort to identify 
transcription factors that bind to the CACCC element, we found that purified 
human Sp1, as well as Sp1 in HeLa nuclear extract, can specifically bind to a 
DNA probe that corresponds to the PTHrP-specific sequence between -94 and -34. 
Gel shift competition studies and DNase I footprinting analyses revealed that 
Sp1 specifically interacts with the CACCC motif. In the presence of Ets1, the 
mobility of the Sp1-specific gel shift complex with the PTHrP DNA decreased. 
DNase I footprint analysis of this gel shift complex showed an extended 
footprint over both the Sp1 and the Ets1 binding site, demonstrating that Sp1 
and Ets1 form a ternary complex with the PTHrP DNA. Cotransfection of an Ets1 
and Sp1 expression vector into Drosophila Schneider cells demonstrated that Sp1 
can functionally cooperate with Ets1 to transactivate the PTHrP promoter. We 
conclude from these data that Ets1 and Sp1 can cooperatively regulate PTHrP P2 
promoter activity.