The rod cGMP-phosphodiesterase beta-subunit promoter is a specific target for Sp4 and is not activated by other Sp proteins or CRX

J Biol Chem. 2002 Jul 19;277(29):25877-83. doi: 10.1074/jbc.M201407200. Epub 2002 Apr 9.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases / genetics*
  • Animals
  • Base Sequence
  • Cyclic Nucleotide Phosphodiesterases, Type 6
  • DNA-Binding Proteins / metabolism
  • Homeodomain Proteins / metabolism*
  • Humans
  • Molecular Sequence Data
  • Phosphoric Diester Hydrolases*
  • Promoter Regions, Genetic*
  • Retinal Rod Photoreceptor Cells / enzymology*
  • Sp4 Transcription Factor
  • TATA-Box Binding Protein
  • Trans-Activators / metabolism*
  • Transcription Factor TFIIB
  • Transcription Factors / metabolism*
  • Transfection
  • Tumor Cells, Cultured
  • Xenopus

Substances

  • DNA-Binding Proteins
  • Homeodomain Proteins
  • SP4 protein, human
  • Sp4 Transcription Factor
  • TATA-Box Binding Protein
  • Trans-Activators
  • Transcription Factor TFIIB
  • Transcription Factors
  • cone rod homeobox protein
  • Phosphoric Diester Hydrolases
  • 3',5'-Cyclic-GMP Phosphodiesterases
  • Cyclic Nucleotide Phosphodiesterases, Type 6
  • PDE6B protein, human