DNA–Binding Domains of Sp4 and Nrl Transcription Factors are Required for Sp4–Nrl Functional Synergy on the Ã|*159*|–PDE Gene Promoter

Abstract

Abstract: : Purpose: The Sp family of transcription factors has been implicated in transcriptional control of numerous genes in a variety of tissues. One member of the Sp family, Sp4, is predominantly expressed in the CNS and retina. We previously reported that Sp4 is involved in transcriptional regulation of multiple photoreceptor–restricted genes. Another transcription factor, Nrl, is essential for rod differentiation and is an important regulator of several rod–restricted genes. Sp4 and Nrl have major roles in transcriptional activation of the rod cGMP–phophodiesterase Ã|*159*|–subunit (Ã|*159*|–PDE) promoter. The purpose of this study was to investigate the molecular mechanisms of Sp4–Nrl synergy on the Ã|*159*|–PDE minimal promoter. In particular, we tested the effect of the mutation in the Sp4–response element, Ã|*159*|/GC, on Sp4– or/and Nrl–mediated activation of the Ã|*159*|–PDE promoter. We also evaluated the functional properties of Sp4 and Nrl domain–deletion mutants for their ability to combinatorially activate the Ã|*159*|–PDE minimal promoter. Methods: Transient transfection and co–transfection assays were performed in 293 human embryonic kidney cells. A luciferase reporter construct containing the Ã|*159*|–PDE minimal promoter (–93/+53) was employed. In order to test the mutual functional effects of the transcription factors of interest, expression plasmids encoding Sp4, Nrl, or their domain–deletion mutants were added to transfection mixtures individually or in combinations. Results: Previously, we showed that Sp4 synergizes with Nrl on the Rhodopsin promoter. Here, we found that Sp4 also synergizes with Nrl on the minimal Ã|*159*|–PDE promoter. Furthermore, mutations in the Ã|*159*|/GC sequence decrease Sp4–mediated activation and abolish Sp4–Nrl synergy. Sp4 also synergizes with the leucine zipper–containing Nrl truncation mutant, DD10. The zinc finger domain is critical for Sp4 transcriptional activity and is necessary for Sp4–Nrl transcriptional synergy. Conclusions: These results further corroborate the functional synergy between Sp4 and Nrl on the Ã|*159*|–PDE promoter. DNA–binding domains of both Sp4 and Nrl are required for Sp4–Nrl functional synergy, which may be mediated by their mutual stabilization of the Sp4/Nrl–DNA complex.

Keywords: gene/expression • photoreceptors • retina