Genes: Structure and Regulation
Functional Analysis of the Rod Photoreceptor cGMP Phosphodiesterase α-Subunit Gene Promoter: Nrl AND Crx ARE REQUIRED FOR FULL TRANSCRIPTIONAL ACTIVITY*

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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.

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d

Recipient of a Research to Prevent Blindness career development award.

f

Present address: University of Ottawa Eye Institute, Ottawa Health Research Institute, Ottawa, Canada.

h

The Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology and a Research to Prevent Blindness Senior Scientific Investigator.

i

Present address: National Brain Research Center, Gurgaon, India.

j

The Harold F. Falls Collegiate Professor.

k

Present address: The Salk Institute, Regulatory Biology, La Jolla, CA 92037.

*

This work was supported in part by National Institutes of Health Grants EY09924 (to S. J. P.), EY11115 (to A. S.), EY12543 (to S. C.), and EY09769 (to D. J. Z.); The Foundation Fighting Blindness; Research to Prevent Blindness; and the University of Alabama Health Services Foundation (to S. J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.