Dissection of the promoter region of the inositol 1,4,5-trisphosphate receptor gene, itr-1, in C. elegans: a molecular basis for cell-specific expression of IP3R isoforms

Abstract

Inositol 1,4,5-trisphosphate receptors in Caenorhabditis elegans are encoded by a single gene, itr-1. This provides a powerful system in which to dissect the mechanisms that control the tissue-specific expression of molecules that determine the specificity of calcium signalling. We first identified the Caenorhabditis briggsae orthologue of itr-1, Cbitr-1. Comparison of the two itr-1 genes revealed that the chromosomal organisation, gene structure and predicted cDNA and protein sequences were all conserved. The conserved gene structure supports the hypothesis that the itr-1 gene has three promoters, each of which gives rise to an alternative mRNA and hence unique protein. To test this and to identify the roles of the three putative promoters (pA, pB and pC) in regulating itr-1 expression we fused each promoter to the green fluorescent protein gene and identified their expression patterns. Introduction of these transgenes into C. elegans identified unique and defined patterns of green fluorescent protein expression directed by each promoter: pA directs expression in the pharyngeal terminal bulb, the rectal epithelial cells and vulva; pB directs expression in the motor neurone PDA, the amphid socket cells and the spermatheca; pC directs expression in the spermathecal valve, uterine sheath cells, pharyngeal isthmus and intestine. Thus tissue-specific expression of itr-1 variants is directed by three promoters and this results in adjacent cells in the same tissue containing different inositol trisphosphate receptor isoforms.Within pA, four short regions (pA-A to pA-D) of sequence conservation between C. elegans and C. briggsae were identified. Deletion analysis demonstrated that the region containing pA-C is required for expression in the terminal bulb and rectal epithelial cells and the region containing pA-D is required for expression in the vulva. pA-C includes sequences similar to the binding sites for transcription factors that have been demonstrated to be important in pharyngeal development and gene expression.