Özet:
In the olfactory system, each olfactory sensory neuron (OSN) typically expresses only one allele of a single olfactory receptor (OR) gene from a vast genomic repertoire. The mechanisms of OR gene choice, however, are not well understood and may include epigenetic modification as well as locus-specific control by cis-regulatory sequences. It has been shown that both, proximal promoter regions and long-range interacting elements contribute to OR gene choice. Here, critical elements regulating expression of a model zebrafish OR gene, OR101-1, were identified and tested functionally using a transient transgenic approach. In particular, the negative regulatory effect of a specific 500 bp sequence (500i), located 2 kb upstream of the OR101-1 gene have been investigated. Using a promoter bashing approach, it was shown that transgenic constructs that included this 500 bp sequence were less effective, both in the number of embryos and OSNs per embryo that expressed a fluorescent reporter transgene. To further analyze the regulatory influence of this sequence, new transgenic constructs were generated by cloning 500i upstream of a strong OR gene promoter and scored for transgene expression in early embryos. Inclusion of the sequence reduced transgene efficiency by 50%. Next, the role of two candidate transcription factor binding sites located within 500i were investigated using site-directed mutagenesis. Deletion of either one or both binding sites from transgenic constructs was sufficient to rescue high levels of transgene expression, suggesting that these sites, and transcription factors binding to these sites, may be responsible for the observed repressive effect. To further elucidate a biological function for this type of regulation, it was tested whether the sequence could act as insulator to shield the OR101-1 promoter from the influence of a nearby enhancer. Indeed, transgenic constructs that included 500i interspersed between a strong enhancer and the OR101-1 promoter resulted in a reduction of transgene-expressing OSNs. Mutation of the suspected transcription factor binding sites partially rescued the enhancer effect. These findings are significant since they functionally demonstrate, for the first time, that OR gene expression is also under control by repressive regulators.
