Structure and upstream region characterization of the human gene encoding rod photoreceptor cGMP phosphodiesterase α-subunit

Abstract

Rod photoreceptor cGMP phosphodiesterase (PDE6) is a three-subunit (a, b, g2) enzyme that functions to reduce intracellular cytoplasmic cGMP levels, an integral feature of the phototransduction cascade of vision. To allow assessment of the potential for defects in the gene encoding the alpha-subunit (PDE6A) to cause visual dysfunction, and to begin to dissect the basis for photoreceptor-specific expression of this gene, we have characterized the structural gene and upstream region. The human PDE6A gene consists of 22 exons spanning about 60 kb with the intron/exon junctions highly conserved in comparison to the mouse and human PDE6B genes. Using ribonuclease protection and primer extension assays, a predominant transcription start point (tsp) was identified 120 bp upstream of the initiator ATG. To begin functional analysis of the PDE6A promoter, approx 4 kb of sequence were determined upstream of the tsp. Comparison of this upstream sequence with an approximately 500 bp sequence upstream of the mouse Pde6a gene revealed five distinct segments of identity all within 100 bp upstream of the human PDE6A tsp. A TATA box adjacent to a photoreceptor-specific RET1-like binding site, an SP1 site, and two novel putative cis-element sequences were found. A consensus initiator element sequence is present at the tsp. Additionally, within a 2.5-kb segment beginning 900 bp upstream of the tsp two Alu, a MIR, an L1, and two MER repetitive elements were found. Electrophoretic mobility shift assays generate a retina-specific bandshift using a 322-bp fragment containing the putative promoter region or a multimer of the RET1-like site. DNA footprinting assays revealed footprints over the primary transcription startpoint and the RET1-like and TATA box regions. These results indicate that a 220-bp segment of the PDE6A gene upstream region is important for tissue-specific expression.

Functional Analysis of the Rod Photoreceptor cGMP Phosphodiesterase α-Subunit Gene Promoter

“…The estimated size of these fragments places the hypersensitive sites immediately upstream of the predicted core promoter region. This result is consistent with an active promoter within the region previously identified in vitro (10).…”

“…The data are consistent, however, with a requirement for defined DNA bending and possible cooperativity of Crx binding. Also consistent with this idea is our previous finding using human retina nuclear extracts in gel shift assays, in which a retina-specific shift was only observed when the entire promoter region was included in the probe (10). The rhodopsin proximal promoter region is composed of a very similar array of cis-elements with an NRE flanked on both sides by Crx binding sites (29), and this promoter also shows weak activity in the absence of an upstream enhancer (30).…”

“…We reported previously that the PDE6A gene promoter is contained within an ϳ4-kb region upstream of the transcription start point, contains several potential cis-elements, and can bind tissue-specific factors in a retinal nuclear extract (10). A DNase I footprinting region was identified within the first 100 upstream nucleotides that was confirmed by the demonstration of a retina-specific mobility shift.…”

“…1 is the portion of the PDE6A core promoter region analyzed in this study. This region contains three potential Crx binding elements (CBE1-3), one site resembling an Nrl/AP1 binding element (NRE/AP1), and one additional segment that is conserved in the upstream regions of both human and mouse (10). To assess the chromatin status of the region around the PDE6A promoter region in vivo, a DNase I hypersensitivity assay was performed with Y79 cells, which transcribe PDE6A mRNA (26).…”

A PDE6A Promoter Fragment Directs Transcription Predominantly in the Photoreceptor

“…We previously identified a short upstream fragment of the PDE6A gene that binds retina-specific transcription factors (18). To determine the tissue specificity directed by this fragment we created a reporter gene construct for introduction into the mouse genome.…”