Sp4 is a human sequence-specific DNA binding protein with structural features similar to those described for the transcription factors Sp1 and Sp3. These three proteins contain two glutamine-rich regions and a highly conserved DNA binding domain composed of three zinc fingers. Consistently, Sp1, Sp3, and Sp4 do have the same DNA binding specificities. In this report, we have embarked on a detailed analysis of the transcriptional properties of Sp4 in direct comparison to Sp1 and Sp3. Cotransfection experiments into Drosophila SL2 cells lacking endogenous Sp factors demonstrate that Sp4 is an activator protein like Sp1. However, in contrast to Sp1, Sp4 is not able to act synergistically through adjacent binding sites. The transactivation function of Sp4 resides, like that of Sp1, in the N-terminal glutamine-rich region. Sp4 can function as a target for the Sp1 activation domains in a superactivation assay, suggesting that the activation domains of Sp1 and Sp4 are functionally related. Furthermore, we show that Sp4-mediated transcriptional activation can be repressed by Sp3. Taken together, our results demonstrate that the transcription factor Sp4 exhibits specific functional properties distinct from Sp1 and Sp3.

The human ABCA2 transporter gene encodes a member of a large family of ATP-binding proteins that transport a variety of macromolecules across biological membranes. We have performed luciferase reporter gene assays with promoter constructs comprising the 5'-flanking region to identify cis-regulatory DNA elements and have mapped the minimal promoter region to 321 bp upstream of the translation start site. We have discovered a functional role for two GC-boxes located in the proximal promoter of the ABCA2 gene that contain overlapping sites for the EGR-1 and Sp1 transcription factors. We observed that oligonucleotides containing overlapping EGR-1/Sp1 sites bind the Sp1, Sp3 and Sp4 transcription factors. When BE(2)-M17 cells were treated with phorbol 12-myristate 13-acetate, we observed inducible expression and binding of the EGR-1 transcription factor to the two GC-boxes. Transfection of Sp1, Sp3 or Sp4 expression constructs into Drosophila S2 induced a dose-dependent increase in transcriptional activation of the ABCA2 promoter, but transfection of EGR-1 alone failed to activate transcription. When increasing amounts of EGR-1 were transfected into the BE(2)-M17 neuroblastoma cells we observed a dose-dependent decrease in expression of the ABCA2 promoter, although expression of the endogenous ABCA2 gene increased following transfection of EGR-1.