Complex interactions with direct repeats of a mitogen-responsive VL30 enhancer

The RVL-3 VL30 enhancer is an LTR-derived triple direct repeat of 35 base pairs that mediates gene induction in response to several different intracellular signaling pathways. Using mobility shift assays, methylation interference and DNase I footprinting, we have investigated the physical interactions between the RVL-3 enhancer and components of nuclear extracts from Rat-1 cells. Each enhancer repeat unit contains a single binding site. Our studies suggest that binding to the double or triple repeat enhancer is cooperative, involving simultaneous occupation of two sites, with a preference for adjacent sites. Binding cooperativity would have implications for the mechanism of gene activation directed from the native VL30 enhancer.