Characterization of the PDE6A Promoter Region in vivo and in Native Chromatin

Abstract

Abstract: : Purpose: Our goal in this study was to define the minimal promoter region of the PDE6A promoter and to demonstrate an open chromatin state of this region in vivo. Methods: Promoter region constructs were analyzed for activity using adenofection in Y79 cells. Individual Crx binding sites were inactivated by PCR with mutagenic primers followed by cassette replacement. Expression constructs for Crx, Nrl and DD10, a truncated NRL competitor, were generated in pcDNA3.1/HisC, pED, and pMT3, respectively. Transactivation by Crx and Nrl was assessed in transient transfection assays in HEK293 cells. In vitro DNaseI footprinting was performed to localize binding sites of Crx. In vivo DNaseI hypersensitivity assays were done with nuclei purified from Y79 and control cells. Results: Promoter assays in Y79 cells show the 300 bp upstream fragment is the most active with successively less activity as construct length increases. Co-transfection of Nrl or Crx and the 300 bp PDE6A promoter construct resulted in a 25-30 fold stimulation of reporter activity in HEK293 cells. In the presence of both Crx and Nrl a 150-fold stimulation of promoter activity was observed. DNaseI footprinting with recombinant Crx protein showed a broad footprint spanning three putative sites within 100 bp upstream of the transcription start point (tsp). Inactivation of site 1 reduced reporter activity by 70 %, and inactivation of site 2 reduced activity by 50 %. Addition of the DD10 construct abolished transactivation of NRL alone and eliminated the synergistic effect observed with Nrl and Crx. The effects of Nrl and Crx were reduced about 20 % with the 1 kb construct. In Y79 cells, addition of the Crx and Nrl constructs increased PDE6A promoter activity 2.5 and 7 fold separately, and 10 fold together. DNAseI hypersensitivity sites indicative of an open chromatin state map to an ~150 bp region of the PDE6A gene only in Y79 cells. Conclusions: Crx appears to bind to at least two sites in the PDE6A promoter that are important for promoter activity. Nrl transactivates the PDE6A gene in the absence of Crx, and acts synergistically with Crx. The native PDE6A promoter defined by DNAseI accessibility assays in intact Y79 nuclei corresponds to the promoter region determined in vitro.

Keywords: gene/expression • photoreceptors • molecular biology