CIS-ACTING AND TRANS-ACTING FACTORS REGULATING TRANSCRIPTION OF THE BGT1 GENE IN RESPONSE TO HYPERTONICITY

We have previously identified a tonicity-responsive enhancer (TonE) in the promoter region of the canine BGT1 gene. TonE mediates hypertonic ity-induced stimulation of transcription. Here, we characterize TonE a nd TonE binding proteins (TonEBPs) to provide a biochemical basis for cloning of the TonEBPs. Mutational analysis applied to both hypertonic ity-induced stimulation of transcription and TonEBP binding reveals th at TonE is 11 base pairs in length, with the consensus sequence of (C/ T)GGAAnnn(C/T)n(C/T). Activity of the TonEBPs increases in response to hypertonicity with a time course similar to that of transcription of the BGT1 gene. Studies with inhibitors indicate that translation, but not transcription, is required for activation of the TonEBPs. Phosphor ylation is required for the stimulation of transcription but not for a ctivation of DNA binding by the TonEBPs. In vivo methylation by dimeth yl sulfate reveals that the TonE site of the BGT1 gene is protected wi th a time course like that of activity of the TonEBPs and activation o f transcription. Ultraviolet cross-linking indicates that the TonEBPs share a DNA binding subunit of 200 kDa.