A class I odorant receptor enhancer shares a functional motif with class II enhancers

In the mouse, 129 functional class I odorant receptor (OR) genes reside in a ~ 3 megabase huge gene cluster on chromosome 7. The J element, a long-range cis -regulatory element governs the singular expression of class I OR genes by exerting its effect over the whole cluster. To elucidate the molecular mechanisms underlying class I-specific enhancer activity of the J element, we analyzed the J element sequence to determine the functional region and essential motif. The 430-bp core J element, that is highly conserved in mammalian species from the platypus to humans, contains a class I-specific conserved motif of AAACTTTTC, multiple homeodomain sites, and a neighboring O/E-like site, as in class II OR-enhancers. A series of transgenic reporter assays demonstrated that the class I-specific motif is not essential, but the 330-bp core J-H/O containing the homeodomain and O/E-like sites is necessary and sufficient for class I-specific enhancer activity. Further motif analysis revealed that one of homeodomain sequence is the Greek Islands composite motif of the adjacent homeodomain and O/E-like sequences, and mutations in the composite motif abolished or severely reduced class I-enhancer activity. Our results demonstrate that class I and class II enhancers share a functional motif for their enhancer activity.