Regulation of parathyroid hormone-related protein (PTHrP) gene expression. Sp1 binds through an inverted CACCC motif and regulates promoter activity in cooperation with Ets1

• Published in: The Journal of biological chemistry 1994-08, Vol.269 (34), p.21428-21434
• Main Authors: Dittmer, J, Gégonne, A, Gitlin, S D, Ghysdael, J, Brady, J N
• Format: Article
• Language: English
• Subjects: Base Sequence; Cells, Cultured; DNA Mutational Analysis; Gene Expression Regulation; Humans; Molecular Sequence Data; Oligonucleotides - metabolism; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic - genetics; Protein Binding; Protein Biosynthesis; Proteins - genetics; Recombinant Fusion Proteins - biosynthesis; Retroviridae Proteins, Oncogenic - metabolism; Sp1 Transcription Factor - metabolism; T-Lymphocytes - cytology; T-Lymphocytes - metabolism; Transcriptional Activation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)31821-5

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.