Functional identification of transcription control sequences of the mouse Crry gene

The mouse C receptor-related gene Crry is expressed by a wide variety of cells. Those sequences required for the transcriptional control of this gene were identified by deletion analysis of regions 5' of the initiating ATG. Fusion of Crry promoter sequences to the reporter gene CAT identified a region approximately 1,500 bp upstream of the transcriptional start site that enhanced transcription of this gene construct. Gel shift and methylation interference assays were performed, and a specific protein-DNA complex was identified within this region. These assays defined a 16-bp sequence 1,642 bp 5' of the initiating ATG that bound a protein in nuclear extracts prepared from all murine cell lines and tissues examined. The methylation interference assay indicated that the core region of the DNA sequence recognized by the protein was GGAA, the common core binding site for the ets family of proto-oncogenes. Oligonucleotides prepared from this sequence with the GGAA sequence did inhibit the DNA/protein complex formation, whereas those with a mutant GGAA sites did not. The minimal site identified by methylation interference was able to up-regulate transcription when placed downstream of a heterologous promoter, whereas the same sequence with an altered GGAA site could not. Thus, this site functions as an enhancer.