THE BZIP TRANSCRIPTION FACTOR NRL STIMULATES RHODOPSIN PROMOTER ACTIVITY IN PRIMARY RETINAL CELL-CULTURES

In vitro DNA binding assays and transient transfection analysis with m onkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfam ily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression, We have now further explore d the role of the NRE and surrounding promoter elements. Using the yea st one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clon es 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, transien t expression studies with primary chick retinal cell cultures were per formed. Deletion and mutation analyses identified two positive regulat ory sequences: one between -40 and -84 base pairs (bp) and another bet ween -84 and -130 bp. Activity of the -40 to -84 region was shown to b e largely due to the NRE, On co-transfection with an NRL expression ve ctor, there were 3-5-fold increases in the activity of rhodopsin promo ter constructs containing an intact NRE but little or no effect with r hodopsin promoters containing a mutated or deleted NRE. Nrl was more e ffective than the related bZIP proteins, c-Fos and c-Jun, in stimulati ng rhodopsin promoter activity. The -84- to -130-bp region acted syner gistically 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 addit ional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the c is-acting response elements and trans-acting factors that regulate pho toreceptor gene expression.