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Originally published In Press as doi:10.1074/jbc.M313589200 on January 15, 2004

J. Biol. Chem., Vol. 279, Issue 14, 13911-13924, April 2, 2004
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Sp2 DNA Binding Activity and trans-Activation Are Negatively Regulated in Mammalian Cells*

K. Scott Moorefield, Sarah J. Fry, and Jonathan M. Horowitz{ddagger}

From the Graduate Program in Genomic Sciences and Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606

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 pM) 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.

Received for publication, December 11, 2003 , and in revised form, January 14, 2004.

* This work was supported by National Institutes of Health Grants CA53248 and GM065405 and National Science Foundation Grant IGERT 9987555. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{ddagger} To whom correspondence should be addressed: Dept. of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh, NC 27606. Tel.: 919-515-4479; Fax: 919-515-3044; E-mail: jon_horowitz{at}ncsu.edu.


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