Initiation of myoblast to brown fat switch by a PRDM 16 – C / EBP-b transcriptional complex

Brown adipose cells are specialized to dissipate chemical energy in the form of heat, as a physiological defence against cold and obesity. PRDM16 (PR domain containing 16) is a 140 kDa zinc finger protein that robustly induces brown fat determination and differentiation. Recent data suggests that brown fat cells arise in vivo from a Myf5positive, myoblastic lineage by the action of PRDM16 (ref. 3); however, the molecular mechanisms responsible for this developmental switch is unclear. Here we show that PRDM16 forms a transcriptional complex with the active form of C/EBP-b (also known as LAP), acting as a critical molecular unit that controls the cell fate switch from myoblastic precursors to brown fat cells. Forced expression of PRDM16 and C/EBP-b is sufficient to induce a fully functional brown fat program in naive fibroblastic cells, including skin fibroblasts from mouse and man. Transplantation of fibroblasts expressing these two factors into mice gives rise to an ectopic fat pad with the morphological and biochemical characteristics of brown fat. Like endogenous brown fat, this synthetic brown fat tissue acts as a sink for glucose uptake, as determined by positron emission tomography with fluorodeoxyglucose. These data indicate that the PRDM16–C/EBP-b complex initiates brown fat formation from myoblastic precursors, and may provide opportunities for the development of new therapeutics for obesity and type-2 diabetes. Because of the importance of brown adipose tissue (BAT) as a natural defence against hypothermia and obesity, and its demonstrated presence in adult humans, understanding its formation in mechanistic detail may open new avenues to the development of new therapeutics for metabolic diseases such as obesity and type-2 diabetes. Several transcriptional regulators have been identified that positively or negatively control BAT development including RB1 (ref. 8), p107 (also known as RBL1), RIP140 (NRIP1) and FOXC2 (ref. 11). Most recently, we have shown that PRDM16, a 140-kDa zinc finger protein, functions as a bidirectional switch in brown fat cell fate by stimulating the development of brown fat cells from white preadipocytes and from Myf5-positive myoblastic precursors in vitro and in vivo. At a molecular level, PRDM16 works as a transcriptional co-regulatory protein by co-activating PPARc (peroxisome proliferator-activated receptor c), which is considered the ‘master’ gene of fat cell differentiation, and this is almost certainly an important event in the conversion of myoblasts to brown adipocytes. However, both isoforms of PPARc are expressed at very low levels in primary and immortalized myoblasts, whereas they are abundantly expressed in white and brown preadipocytes (Supplementary Fig. 1). Hence, it is very likely that PRDM16 initiates the process of myoblast to brown fat conversion by complexing with other DNA-binding factors, well before the coactivation of PPARc. We therefore devised a strategy to address this, as illustrated in Fig. 1a. In brief, we performed proteomic analyses of transcriptional complexes formed with wild-type PRDM16 or different mutant alleles that were differentiation-competent or -incompetent. Transcription factors that co-purified preferentially with differentiationcompetent PRDM16 proteins were identified; their expression in white and brown fat was then analysed and compared to that of PRDM16. Subsequently, we examined their function in the process of myoblast to brown fat conversion through PRDM16. As shown in Fig. 1b, wild type PRDM16 and a mutant protein lacking the PR (PRD1-BF1-RIZ1 homologous) domain (DPR; amino acids 91–223) that shares homology to the SET chromatin remodelling domain, induced brown fat cell differentiation from myoblasts. In