This is a new RO1 application from a senior investigator that requests 5 years of support to identify genes that control the induction of brown adipocytes in white fat tissue. Prior work, much of it based on seminal contributions of the PI, indicates that brown fat thermogenesis mediated by Uncoupling protein 1 can have a significant impact on energy homeostasis, and that activation of this program can proceed through previously existing brown fat but also, importantly, by the development of new brown fat within white adipose tissue of adult animals. Using Ucp1 mRNA levels as a marker for the extent of brown fat induction in retroperitoneal fat (which is normally white fat and so does not express Ucp1), the PI proposes to further characterize and identify 4 or 5 so-called Iba genes that have been identified in preliminary QTL and RI studies of B6 vs. A/J mice. Each of the QTLs has been localized to a 10 - 15 cM interval;
in Aim 1, Iba genetic architecture will be investigated by constructing B6.A and A.B6 congenic lines for each of the loci, then measuring the effect of those QTLs by themselves and in 16 different combinations. For QTLs whose effect remains robust when examined in isolation as a congenic line, further genetic crosses will be carried out in Aim 2 to narrow the region; in addition, corresponding strains from a genome-wide panel of B6.CAST congenics will be evaluated for the Iba phenotype and, if positive, used in parallel with the B6 vs. A/J cross.
In Aim 3, BAC contigs will be generated for loci whose position can be narrowed to 1 - 2 cM (the PI suggests that Iba4 on chromosome 19 meets this criteria), and candidate genes will be identified by direct sequencing and reference to B6 and A/J retroperitoneal fat cDNA libraries the PI proposes to construct. In parallel (Aim 4), SAGE analysis will be undertaken using retroperitoneal fat RNA from the congenic lines. Finally (Aim 5), candidate cDNAs or genes will be tested by complementation in a cell culture system based on preadipocytes or BAC transgenesis using B6.A congenics.
