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 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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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3',5'-Cyclic-GMP Phosphodiesterases / chemistry*
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Adenoviridae / genetics
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Animals
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Base Sequence
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Basic-Leucine Zipper Transcription Factors
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Binding Sites
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Calcium Phosphates / metabolism
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Cell Line
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Cell Nucleus / metabolism
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Cloning, Molecular
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Cyclic Nucleotide Phosphodiesterases, Type 6
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DNA / metabolism
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DNA Mutational Analysis
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DNA-Binding Proteins / physiology*
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Deoxyribonuclease I / metabolism
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Dose-Response Relationship, Drug
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Eye Proteins / physiology*
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Genes, Dominant
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Glutathione Transferase / metabolism
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Homeodomain Proteins / physiology*
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Humans
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Luciferases / metabolism
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Mice
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Mice, Knockout
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Molecular Sequence Data
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Mutation
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Plasmids / metabolism
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Polymerase Chain Reaction
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Promoter Regions, Genetic*
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Protein Binding
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Retina / metabolism
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Retinal Rod Photoreceptor Cells / enzymology*
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Reverse Transcriptase Polymerase Chain Reaction
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Time Factors
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Trans-Activators / physiology*
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Transcription, Genetic*
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Transcriptional Activation
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Transfection
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beta-Galactosidase / metabolism
Substances
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Basic-Leucine Zipper Transcription Factors
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Calcium Phosphates
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DNA-Binding Proteins
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Eye Proteins
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Homeodomain Proteins
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NRL protein, human
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Nrl protein, mouse
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Trans-Activators
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cone rod homeobox protein
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DNA
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calcium phosphate
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Luciferases
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Glutathione Transferase
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Deoxyribonuclease I
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3',5'-Cyclic-GMP Phosphodiesterases
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Cyclic Nucleotide Phosphodiesterases, Type 6
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PDE6A protein, human
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Pde6a protein, mouse
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beta-Galactosidase