The -subunit of cGMP-phosphodiesterase (-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. -PDE promoter deletions showed that the ؊45/؊23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical -PDE transcriptional regulator in vivo. Interestingly, although the /GC element (؊59/؊49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced -PDE promoter activity. Thus, the Sp4-mediated differential activation of the -PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the -PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich /TA sequence located within the ؊45/؊23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity.One of the key components of the phototransduction cascade that takes place in rod photoreceptors is the heterotetrameric (␣␥ 2 ) cGMP-phosphodiesterase (1). The gene encoding the -subunit of the human enzyme (-PDE) 1 has been well characterized and consists of 22 exons encompassing ϳ43 kb of genomic DNA (2). Genetic defects in this gene have been linked to retinal degeneration in several animal species and human (3-9). There is increasing evidence that abnormalities in transcriptional regulatory components of different genes contribute significantly to or directly cause pathological phenotypes in the retina (10 -13). Therefore, further studies on the transcriptional regulation of rod-specific -PDE gene will identify additional genes important for retinal function and structural integrity and will ultimately help to establish the molecular mechanisms crucial for retina-specific expression of this and perhaps some other genes.We recently reported our initial results on the transcriptional control mechanisms that take place in the human -PDE 5Ј-flanking region (14). Mutational analysis of the -PDE promoter tested both in vitro and ex vivo, and confirmed by the generation of transgenic Xenopus expressing mutant -PDE promoter/green fluorescent protein fusion constructs in vivo, revealed a minimal promoter region, from Ϫ93 to ϩ53, that supports high levels of rod-specific transcription (14). Two enhancer elements were localized within this minimal promoter, Ap1/NRE and /GC, that interact with nuclear factors and activate transcription from the -PDE promoter.To continue the systematic analysis of the -PDE promoter...