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

被引:46
作者
Pittler, SJ
Zhang, YW
Chen, SM
Mears, AJ
Zack, DJ
Ren, ZY
Swain, PK
Yao, SX
Swaroop, A
White, JB
机构
[1] Univ Alabama Birmingham, Sch Optometry, Vis Sci Res Ctr, Dept Physiol Opt, Birmingham, AL 35294 USA
[2] Washington Univ, Sch Med, Dept Ophthalmol & Visual Sci, St Louis, MO 63110 USA
[3] Univ Michigan, Kellogg Eye Ctr, Dept Ophthalmol, Ann Arbor, MI 48105 USA
[4] Univ Michigan, Kellogg Eye Ctr, Dept Human Genet, Ann Arbor, MI 48105 USA
[5] Johns Hopkins Med Sch, Dept Ophthalmol, Baltimore, MD 21287 USA
[6] Johns Hopkins Med Sch, Dept Mol Biol & Genet, Baltimore, MD 21287 USA
[7] Johns Hopkins Med Sch, Dept Neurosci, Baltimore, MD 21287 USA
关键词
D O I
10.1074/jbc.M401864200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:19800 / 19807
页数:8
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