Sp2 DNA Binding Activity and trans-Activation Are Negatively Regulated in Mammalian Cells

• Published in: The Journal of biological chemistry Vol. 279; no. 14; pp. 13911 - 13924
• Main Authors: Moorefield, K Scott, Fry, Sarah J, Horowitz, Jonathan M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 02.04.2004
• Subjects: Animals; Base Sequence; Consensus Sequence; COS Cells; Cricetinae; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Epithelial Cells - cytology; Epithelial Cells - physiology; Humans; Male; Mammals; Mice; Promoter Regions, Genetic - physiology; Prostate - cytology; Prostate-Specific Antigen - genetics; Protein Structure, Tertiary; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sp1 Transcription Factor - chemistry; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - metabolism; Sp2 Transcription Factor; Sp3 Transcription Factor; Tetrahydrofolate Dehydrogenase - genetics; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M313589200

Previous studies have indicated that Sp2 binds poorly to GC-rich sequences bound by Sp1 and Sp3, and further functional analyses of Sp2 have been limited. To study Sp2-mediated transcription, we employed a PCR-based protocol to determine the Sp2 consensus DNA-binding sequence (5′-GGGCGGGAC-3′) and performed kinetic experiments to show that Sp2 binds this consensus sequence with high affinity (225 p m ) in vitro . To determine the functional consequence of Sp2 interaction with this sequence in vivo , we transformed well characterized Sp-binding sites within the dihydrofolate reductase (DHFR) promoter to consensus Sp2-binding sites. Incorporation of Sp2-binding sites within the DHFR promoter increased Sp2-mediated trans -activation in transient co-transfection experiments but also revealed Sp2 to be a relatively weak trans -activator with little or no capacity for additive or synergistic trans -activation. Using chimeric molecules prepared with portions of Sp1 and Sp2 and the human prostate-specific antigen promoter, we show that Sp2 DNA binding activity and trans -activation are negatively regulated in mammalian cells. Taken together, our data indicate that Sp2 is functionally distinct relative to other Sp family members and suggest that Sp2 may play a unique role in cell physiology.