Abstract
In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between -40 and -84 base pairs (bp) and another between -84 and -130 bp. Activity of the -40 to -84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The -84- to -130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.
Highlights
Rhodopsin is the visual pigment of vertebrate rods, and its activation by light initiates the phototransduction process [1]
To identify transcription factors that can bind to the rhodopsin Nrl response element (NRE) and its flanking DNA in vivo, in the context of chromatin structure, we used the yeast one-hybrid system [19, 41, 42]
Chick retinal cells were transfected with a series of luciferase fusion constructs containing DNA from the region upstream of the bovine and murine rhodopsin. (The nomenclature used for reporter constructs includes abbreviations for the species and gene name, followed by a number that refers to the position of the 5Ј-end of the construct relative to the mRNA start site.) The bRho-2174, bRho-225, bRho-176, mRho-4907, mRho-1609, mRho-1488, and mRho-270 constructs demonstrated similar levels of activity, ranging between 66 and 107 relative light units (Fig. 3A). These results demonstrate that the bovine and murine rhodopsin proximal promoter regions, consisting of less than 300 bp of upstream DNA, are as active in chick retinal cultures as longer constructs containing the rhodopsin enhancer region (RER). (The RER spans the region from Ϫ2044 to Ϫ1943 and from Ϫ1575 to Ϫ1477 bp in the cow and mouse genes, respectively [9].)
Summary
Yeast One-hybrid Screening—A retinal cDNA fusion library for onehybrid screening was generated from poly(A)ϩ bovine retinal RNA. Preparation of Constructs—Reporter constructs were generated by cloning appropriate DNA fragments into the multiple cloning site of the pGL2-basic luciferase-containing plasmid (Promega, Madison, WI). The calcium phosphate procedure was optimized with respect to the amount of reporter and carrier DNA, the volume of calcium phosphate precipitate, cell density, the number of days in vitro prior to transfection, and the interval between transfection and harvesting (data not shown). These studies resulted in the protocol described below. The resulting -galactosidase values were used to correct the luciferase values for transfection efficiency
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