Transcription factor FBI-1 acts as a dual regulator in adipogenesis by coordinated regulation of cyclin-A and E2F-4

Aims: Generation of new adipocytes plays a major role in the development of obesity. We previously have shown that transcriptional repressor FBI-1 exerts a dual effect in the process of adipogenesis by inhibiting proliferation and promoting differentiation of preadipocytes. The aim of the present study was to identify FBI-1 regulated molecular effectors which could account for these effects.

Methods: In order to investigate the effect of FBI-1 on human cyclin A and E2F-4 promoter activity, dual-luciferase assays as well as EMSA and ChIP assays were performed in pEGFP-FBI-1 overexpressing and control cells. For expression profiling of potential FBI-1 co-repressors during adipogenesis, 3T3-L1 cells were differentiated into mature fat cells and HDAC-1, HDAC-2 and Sin3A levels were examined by western blotting at different time points of the differentiation process. Finally, using co-immunoprecipitation studies the interaction of pEGFP-FBI-1 and co-repressors was investigated.

Results: Overexpressing FBI-1 in preadipocytes resulted in reduced expression of the cell cycle regulator cyclin A, which may explain FBI-1 induced inhibition of proliferation. Interestingly, FBI-1 repressed cyclin A promoter activity through an indirect mechanism that did not involve direct binding of FBI-1 to the promoter sequence, but rather FBI-1 inhibition of transcriptional activator Sp1 binding to a regulatory element at -452 to -443. We also show that FBI-1 promotes terminal preadipocyte differentiation through a mechanism involving decreased levels of expression of the PPARγ inhibitor E2F-4. FBI-1 significantly reduced E2F-4 promoter activity. Contrary to cyclin A, we found FBI-1 induced repression of E2F-4 is mediated by a direct mechanism via a FBI-1 regulatory element at -11 to -5. Since function of transcriptional repressors normally depends on the presence of regulatory co-factors we also performed expression profiling of potential FBI-1 co-repressors throughout adipogenesis. In these experiments Sin3A and HDAC-1 showed a similar expression pattern compared to FBI-1. Strikingly, co-immunoprecipitation studies revealed that FBI-1 binds Sin3A and HDAC-1 to form a repressor complex. Furthermore, by mutational analysis the aminoterminal POZ domain of FBI-1 was found to be important for Sin3A and HDAC-1 binding.

Conclusions: Taken together, FBI-1 is the first transcriptional repressor shown to act as a dual regulator in adipogenesis exerting repressor activities on target genes by both, direct and indirect mechanisms.