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

Steven J. Pittler, Youwen Zhang, Shiming Chen, Alan J. Mears, Donald J. Zack, Zhiyong Ren, Prabodh K. Swain, Suxia Yao, Anand Swaroop, J. Brandon White

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)19800-19807
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number19
DOIs
StatePublished - May 7 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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