Transgenic analysis of the Zebrafish OR101-1 gene promoter

Abstract

The repertoire of olfactory receptors constitutes the largest gene families in humans and in other vertebrates. Typically, olfactory sensory neurons only express a single member of the large genomic repertoire and only one allele of it. The mechanisms by which olfactory sensory neurons choose to express an olfactory receptor gene for expression and by which other receptors are prevented from being expressed in the same cell are largely unknown. It was shown that translation of the olfactory receptor protein and accumulation in the cell membrane somehow prevents expression of other receptors. However, far less is known about how a sensory neuron initiates expression of a receptor. Regulatory elements located in distal or proximal regions of the receptor coding sequences might affect gene expression, either by selecting a single receptor gene from a larger gene cluster and activating its expression, or by the interaction of specific transcription factors with proximal promoter regions of a gene. Here studies on the transcriptional regulation of a model olfactory receptor, OR101-1, are presented in zebrafish. Four complementary approaches were used in this study to gain insight into OR101-1 gene expression:a BAC transgenesic approachwas utilized to demonstrate the OR101-1 expression profile, 5’-RLM RACE was performed to identify the TSS of the OR101-1 gene, and a promoter bashing approachwas employed to pinpoint specific sequences that control OR101-1 expression in a proximal promoter assay. To complement these studies, basic bioinformatic analysiswas performedon sequences that affect OR101-1 expression to determine candidate binding sites located within the OR101-1 promoter sequence. By using an array of different transgenic constructs and scoring the efficiencies of their expression in a transient transgenic assay, a 500bp region could be identified that is located between 2kb– 2.5kb upstream of the OR101-1 coding sequence, which, when present in transgenic constructs, decreases the efficiency of expression. Partial deletion of either the 5’- or 3’-half of this sequence had intermediate effects, suggesting that multiple binding sites for negative regulators might be present. Bioinformatics reveals that the candidate inhibitory sequence contains two occurrences of a zbtb binding motif, located in either half of the sequence. The results suggest that olfactory receptor gene expression is also controlled by, as of yet undefined, inhibitory mechanisms that might have important implications for the specificity of receptor gene expression.