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

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 alpha-subunit. In vivo DNase I hypersensitivity assays revealed two sites immediately upstream of the PDE6A core p...

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Veröffentlicht in:The Journal of biological chemistry 2004-05, Vol.279 (19), p.19800-19807
Hauptverfasser: Pittler, Steven J, Zhang, Youwen, Chen, Shiming, Mears, Alan J, Zack, Donald J, Ren, Zhiyong, Swain, Prabodh K, Yao, Suxia, Swaroop, Anand, White, J Brandon
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Sprache:eng
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Zusammenfassung: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 alpha-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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M401864200