title: The rod cGMP-phosphodiesterase beta-subunit promoter is a specific target for Sp4 and is not activated by other Sp proteins or CRX.
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.
authors.affiliation: Jules Stein Eye Institute, Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 90095, USA.
authors.forename: Leonid E
authors.initials: LE
authors.lastname: Lerner
authors.forename: Yekaterina E
authors.initials: YE
authors.lastname: Gribanova
authors.forename: Leigh
authors.initials: L
authors.lastname: Whitaker
authors.forename: Barry E
authors.initials: BE
authors.lastname: Knox
authors.forename: Debora B
authors.initials: DB
authors.lastname: Farber
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004268:DNA-Binding Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases; D011401:Promoter Regions, Genetic; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D051706:Sp4 Transcription Factor; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000378:metabolism*; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014981:Xenopus
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C497124:SP4 protein, human; D051706:Sp4 Transcription Factor; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; C109288:cone rod homeobox protein; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517431:PDE6B protein, human