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 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.
Pittler
Steven J
SJ
Department of Physiological Optics, Vision Science Research Center, School of Optometry, University of Alabama at Birmingham, 924 18th Street S., Birmingham, AL 35294, USA. pittler@uab.edu
Zhang
Youwen
Y
Chen
Shiming
S
Mears
Alan J
AJ
Zack
Donald J
DJ
Ren
Zhiyong
Z
Swain
Prabodh K
PK
Yao
Suxia
S
Swaroop
Anand
A
White
J Brandon
JB
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D002130:Calcium Phosphates / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000502:physiology*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins / Q000502:physiology*; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012160:Retina / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015534:Trans-Activators / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D050976:Basic-Leucine Zipper Transcription Factors; D002130:Calcium Phosphates; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; C490037:NRL protein, human; C084884:Nrl protein, mouse; D015534:Trans-Activators; C109288:cone rod homeobox protein; D004247:DNA; C020243:calcium phosphate; D008156:Luciferases; D005982:Glutathione Transferase; D003850:Deoxyribonuclease I; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C410522:PDE6A protein, human; C498747:Pde6a protein, mouse; D001616:beta-Galactosidase