ABSTRACTThecis-acting DNA element known as the E box (consensus sequence CAxxTG) plays an important role in the transcription of a number of cell-specifically expressed genes. The rat insulin I gene, for example, contains two such sequences (IEB1 and IEB2) that are recognized specifically by a characteristic β cell nuclear factor insulin enhancer factor 1 (IEF1). To define the role of these elements better, we tested for cooperative interactions between the IEB sequences. Transfection experiments were performed with a series of plasmids containing the elements separated by different distances. Transcriptional activityin vivois only modestly affected (less than two-fold) when the distances between the IEB elements are changed by a half-integral number of double-helical turns. Surprisingly, plasmids bearing four and six copies of the IEB motif showed sharply reduced activity as compared to those with two copies.In vitroDNA-binding studies revealed that this effect was not due to inability of IEF1 to bind to multiple copies of IEB. Moreover, multiple copies of the IEB sequence were able to inhibit activity of acis-linked Moloney sarcoma virus (MSV) or insulin enhancer upon transfection to β cells but not to other cell types. The above data are consistent with the view that β cells contain a cell-specific repressor molecule capable of binding to multiple copies of IEB and thereby inhibiting transcription. This interpretation was further strengthened byin vivocompetition andtrans-activation experiments. The β-cell-specific repressor activity identified by these studies may play an important role in mediating gene expression in insulin-producing cells, perhaps by regulating the access of helix-loop-helix transcription factors to E-box sequence eleme
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