Functional analysis of the rod photoreceptor cGMP phosphodiesterase α-subunit gene promoter: Nrl and Crx are required for full transcriptional activity

Academic Article

Abstract

  • To understand the factors controlling expression of the cGMP phosphodiesterase type 6 (PDE6) genes, we have characterized the promoter of the human PDE6A gene that encodes the catalytic α-subunit. In vivo DNase I hypersensitivity assays revealed two sites immediately upstream of the PDE6A core promoter region. Transient transfection assay in Y79 cells of constructs containing varying lengths of the promoter region showed a decrease in promoter activity with increasing length. The most active segment contained a 177-bp upstream sequence including apparent Crx and Nrl transcription factor binding sites. Both Crx and Nrl transactivated the PDE6A promoter in HEK293 cells and showed a >100-fold increase when coexpressed. Coexpression of a dominant negative inhibitor of Nrl abolished Nrl transactivation but had no effect on Crx. DNase I footprinting assays identified three potential Crx binding sites within a 55-bp segment beginning 29 bp upstream of the transcription start point. Mutation of two of these sites reduced reporter gene activity by as much as 69%. Gel shifts showed that all three Crx sites required a TAAT sequence for efficient binding. Consistent with a requirement for Crx and Nrl in Pde6a promoter activity, Pde6a mRNA is reduced by 87% in the retina of Crx -/- mice and is undetectable in Nrl-/- mice at postnatal day 10. These results establish that both Nrl and Crx are required for full transcriptional activity of the PDE6A gene.
  • Authors

    Published In

    Digital Object Identifier (doi)

    Author List

  • Pittler SJ; Zhang Y; Chen S; Mears AJ; Zack DJ; Ren Z; Swain PK; Yao S; Swaroop A; White JB
  • Start Page

  • 19800
  • End Page

  • 19807
  • Volume

  • 279
  • Issue

  • 19