Transcriptional regulation of the mouse uncoupling protein-2 gene -: Double E-Box motif is required for peroxisome proliferator-activated receptor-γ-dependent activation

Uncoupling protein-2 (UCP2) is present in many tissues with relevance to fuel metabolism, and its expression is increased in fat and muscle in response to elevated circulating free fatty acids resulting from fasting and high fat feeding. We proposed a role for peroxisome proliferator-activated receptor-gamma (PPAR gamma) as a mediator of these physiological changes in UCP2, because thiazolidinediones also increase expression of UCP2 in these cell types (1), To determine the molecular basis for this regulation, we isolated the 7.3-kilobase promoter region of the mouse UCP2 gene. The -7.3-kilobase/+12-base pair fragment activates transcription of a reporter gene by 50-100-fold. Deletion and point mutation analysis, coupled with gel shift assays, indicate the presence of a 43-base pair enhancer (-86/-44) that is responsible for the majority of both basal and PPAR gamma -dependent transcriptional activity. The distal (-86/-76) part of the enhancer specifically binds Sp1, Sp2, and Sp3 and is indistinguishable from a consensus Sp1 element in competition experiments. Point mutation in this sequence reduces basal activity by 75%. A second region (-74/-66) is identical to the sterol response element consensus and specifically binds ADD1/SREBP1. However, deletion of this sequence does not affect basal transcriptional activity or the response to PPAR gamma, The proximal portion of the enhancer contains a direct repeat of two E-Box motifs, which contributes most strongly to basal and PPAR gamma -dependent transcription of the UCP2 promoter. Deletion of this region results in a 10-20-fold reduction of transcriptional activity and complete loss of PPAR gamma responsiveness. Point mutations in either E-Box, but not in the spacer region between them, eliminate the stimulatory response to PPAR gamma, However, gel shift assays show that PPAR gamma does not bind to this region. Taken together, these data indicate that PPAR gamma activates the UCP2 gene indirectly by altering the activity or expression of other transcription factors that bind to the UCP2 promoter.