Sp2 DNA binding activity and trans-activation are negatively regulated in mammalian cells.
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.
Moorefield
K Scott
KS
Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.
Fry
Sarah J
SJ
Horowitz
Jonathan M
JM
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D016384:Consensus Sequence; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D008297:Male; D008322:Mammals; D051379:Mice; D011401:Promoter Regions, Genetic / Q000502:physiology; D011467:Prostate / Q000166:cytology; D017430:Prostate-Specific Antigen / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051704:Sp2 Transcription Factor; D051705:Sp3 Transcription Factor; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C497119:SP2 protein, human; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051704:Sp2 Transcription Factor; D051705:Sp3 Transcription Factor; D013762:Tetrahydrofolate Dehydrogenase; D017430:Prostate-Specific Antigen