In vivo characterization of the OR101-1 gene proximal promoter region in zebrafish

Abstract

In vertebrates, each olfactory sensory neuron (OSN) of the nose expresses only a single allele of a single molecular chemoreceptor gene from a large genomic repertoire. This monogenic mode of olfactory receptor (OR) expression in OSNs is critical for the sense of smell because it determines the sensitivity of OSNs to odorants and instructs OSN axons to establish appropriate synaptic connections in the brain. However, the molecular mechanisms regulating OR expression are not fully understood. Over the past five years a principle mechanism to explain monogenic OR expression has emerged that is based on LSD1-mediated changes in the epigenetic signatures of OR gene loci. In this model, LSD1 would modify repressive histone marks with low probability to ensure expression of only a single OR per OSN. Yet, not all ORs are expressed with the same probability and / or within the same spatial domain of the olfactory tissue, suggesting that LSD1 is somehow ‘guided’ to O gene loci in a iased fashion. Two types of OR locus-biased control mechanisms were proposed so far; long-range control by distant cis-acting locus control regions and short-range control by proximal promoter elements. Interestingly, both mechanisms share identical families of homeodomain (Lhx2/Emx2) and Olf-1/Ebf-1 transcription factors. To better understand the contribution of proximal promoter sequences in OR gene expression, the regulation of the zebrafish OR101-1 gene was studied by transient transgenic promoter assays. The proximal 1.2 kb of sequence upstream of the OR101-1 coding region drives expression of fluorescence reporter proteins in zebrafish OSNs with high efficiency. To pinpoint positive regulatory sites within this sequence, a series of related transgenic constructs was generated in which specific sequences were mutated or deleted. The OR101-1 gene promoter appears to be rather compact. Only 212 bp ups. of OR101-1 TSS are sufficient to maintain high efficiency of transgene expression. Sites included in this sequence and resembling an O/E-like binding motif appear not to contribute to expression. Interestingly, however, removal of a 345 bp intron resulted in a 50% loss of efficiency of the promoter construct, though it remains unanswered whether an included O/E-like site or splicing per se contribute to expression.