The Rod cGMP-phosphodiesterase β-Subunit Promoter Is a Specific Target for Sp4 and Is Not Activated by Other Sp Proteins or CRX

Debora B. Farber,Leigh Whitaker,Leonid E. Lerner,Yekaterina E. Gribanova,Barry E. Knox

doi:10.1074/jbc.m201407200

Debora B. Farber, Leigh Whitaker + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m201407200

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Abstract

The beta-subunit of cGMP-phosphodiesterase (beta-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. beta-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 beta-PDE transcriptional regulator in vivo. Interestingly, although the beta/GC element (-59/-49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced beta-PDE promoter activity. Thus, the Sp4-mediated differential activation of the beta-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 beta-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 beta/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.

Highlights

One of the key components of the phototransduction cascade that takes place in rod photoreceptors is the heterotetrameric (␣␤␥2) cGMP-phosphodiesterase [1]
The presence of a consensus Crx-response element (CRE) sequence in the minimal rodspecific ␤-PDE promoter prompted us to test whether Crx, a member of the Otx family of homeodomain-containing transcription factors, is involved in transcriptional regulation of the ␤-PDE gene
The interactions between the ␤-PDE promoter and the basal transcription factors were not completely disrupted by limited nucleotide substitutions in this region, which may be related to the complex, low affinity, basal transcription factor-promoter interactions over extended core promoter sequences described on other promoters [17, 18]

Summary

Introduction

One of the key components of the phototransduction cascade that takes place in rod photoreceptors is the heterotetrameric (␣␤␥2) cGMP-phosphodiesterase [1]. To control for specificity of retinal cell type expression, all ␤-PDE promoter constructs were transfected in dissected Xenopus embryo trunks that contain many cell types [14, 23] and showed no activity (data not shown).

Results

Conclusion