The role of Tip60 in adipogenesis

Abstract

Besides providing insulation and protection against mechanical stress, white adipose tissue (WAT) has long been recognized as a storage depot for excess energy. WAT also releases a wide range of adipokines, which for example regulate immune responses, blood pressure and reproduction. Expansion of WAT, as seen during the development of increased bodyweight and obesity, involves an increase in adipocyte size and the formation of new adipocytes from precursor cells (adipogenesis). Since WAT is increasingly being recognized as a key regulator of whole-body energy homeostasis and consequently as a prime therapeutic target for metabolic syndrome, adipocyte differentiation and biology are under intensive study. A complex cascade of transcriptional events regulates adipogenesis. Recently, we identified the coregulator protein Tip60 as an essential player in adipogenesis. In this thesis we investigated the exact role of the Tip60 in adipogenesis. Previous studies had indicated that Tip60 protein expression but not mRNA expression was upregulated during adipocyte differentiation. We therefore examined whether the protein is normally degraded by the ubiquitin-proteasome system (UPS) and protected from degradation by deubiquitination during adipogenesis. USP7, a dominant deubiquitinating enzyme (DUB) in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinated Tip60 both in intact cells and in vitro, and increased Tip60 protein levels. Inhibition of USP7 expression and activity decreased adipogenesis. Transcriptome analysis revealed several cell cycle genes to be co-regulated by both Tip60 and USP7. Knock down of either factor resulted in impaired mitotic clonal expansion, an early step in adipogenesis. These results therefore reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis. We also tried to identify the ubiquitin E3 ligase responsible for Tip60 ubiquitination in adipocytes. In contrast to the Tip60 protein, expression of the candidate E3 ligase mdm2 was relatively constant during 3T3-L1 adipogenesis. In mdm2-negative MEFs, Tip60 could still be ubiquitinated. These findings indicate that E3 ubiquitin ligases other than mdm2 are probably responsible for Tip60 degradation during early adipogenesis. Next we investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. As homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used, and no major metabolic effects were observed. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice. Finally we developed a novel method to identify adipogenic factors using RNAi-mediated knock down libraries (siRNA libraries), based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting.