A study of thecAMP-response elements of the mouse uncoupling protein 1 enhancer

Uncoupling protein 1 (UCP1) is a critical mediator in brown adipose tissue (BAT) thermogenesis. UCP1, which is found only in BAT, is expressed in response to sympathetic nervous system (SNS) stimulation. This work is a critical investigation of the 220 by mouse UCP1 enhancer and the two cAMP response elements-3 and -2 (CRE-3 and CRE-2) contained within it.; Cell culture analysis of these two CREs demonstrates that the two elements are not functionally equivalent. CRE-3 plays a critical role in norepinephrine stimulated enhancer function. CRE-2 plays a lessor role of important to stimulated enhancer function, but appears to be important for basal level activity of the enhancer. Both CRE-3 and CRE-2 were shown to bind CREB-like proteins by EMSA, however only CRE-3, when present in tandem, had norepinephrine stimulated enhancing activity in the brown adipocytes cell line, HIB 1B Clone 3.; A smaller fragment of the mouse UCP1 enhancer, the 5′-120 bp region, was also investigated in vivo for tissue specificity and thermoregulation of a reporter gene. Out of the five founders identified only one animal line had BAT-specific expression of the reporter gene. However, reporter gene expression was not exclusively in the BAT. All animal lines expressed the reporter gene in the testis. These results may indicate that the 5′-120 bp reporter gene construct lacks a tissue specific repressive region.; An in vivo analysis of the CREs contained within the 220 bp enhancer was also conducted to determine their role in BAT-specific expression and thermoregulation of a reporter gene. The mutant enhancers were still capable of BAT-specific expression; however the expression was lower and more variable than in the 220 bp enhancer transgenic mice (Tai 1998). The effect of mutation on the enhancer ability to thermoregulate was also examined. It was demonstrated neither the CRE-3 mutation nor the double CRE mutation had any effect on thermoregulation. These results demonstrate that the enhancer is composed of multiple response element which in the absence of the CREs are sufficient for thermoregulation of reporter gene.