Several factors contribute to the onset of obesity including molecular imbalances in energy metabolism. Mammals in their native environments are protected from obesity and the cold by adaptive thermogenic survival mechanisms. The objective of this research is to further delineate the relationship between thermogenesis, energy imbalance and obesity by elucidating the control mechanisms underlying the activation of thermogenesis in brown adipose tissue. The major effector of thermogenesis is the brown adipose tissue specific mitochondrial uncoupling protein. My working hypothesis is that several DNA-binding proteins control the onset of thermogenesis following cold exposure by regulating the rate of mitochondrial uncoupling protein gene (Ucp) transcription in brown adipose tissue. The focus of SPECIFIC AIM I will be to elucidate the binding domains for the cold-inducible DNA-binding proteins which induce Ucp expression in brown adipose tissue. Three transgenic constructs will be microinjected to delineate the 5' Ucp regulatory domain to within 500 bp. A rigorous search for protein-DNA interactions will then be initiated within this regulatory domain by DNA footprinting and gel shift analysis.
In SPECIFIC AIM II, the functional significance of these binding domains will be evaluated using in vitro transcription assays with brown adipose tissue nuclear extracts obtained from cold and thermoneutral acclimatized mice. The final functional test for a given protein-DNA binding domain will again be performed using transgenic mice. The goal of SPECIFIC AIM III is to isolate cDNA's for the transcription factors which participate in the cold-induced activation of Ucp using recognition site oligonucleotide probes, and begin a systematic analysis into the regulation of the transcription factors themselves. By elucidating the signalling mechanisms controlling thermogenesis and energy expenditure, this research will result in a better understanding of defective energy expenditure, energy imbalance and obesity.
