GEN-AU Gold III (Genomics of Lipid-associated Disorders III); C3: Identification and characterization of genes involved in fat cell development

Aim 1: Generation and characterization of adipose tissue-specific APMAP transgenic mice. In a transcriptomics approach we recently found APMAP to be highly up-regulated during 3T3-L1 differentiation and to play an important role in adipogenesis in vitro [Strauss et al., manuscript in preparation]. To elucidate the physiological function of this gene we will generate and characterize adipose tissue-specific transgenic mice will be generated using a plasmid containing the 5.4-kb mouse aP2 promoter that will be fused to the 1.25 kb APMAP cDNA, linearized, purified, and microinjected into the fertilized pronuclei of female mice that had been mated before. Furthermore, the human in vivo function and role of APMAP will be studied by investigation of the genetic variability within obesity-related phenotypes in human association studies (C13/Kronenberg). Aim 2: Functional characterization of genes differentially regulated in fat cell development. Using microarray analysis described above, a number of genes with yet unknown functions displaying significant differential regulation during fat cell development was found, such as APMAP, RIKEN gene 653040D17 (Arxes), 24100004L22, and 4930566A11. First, APMAP will be studied in more detail in vitro. Hydrolase assays (due to the prediction of sequence analysis by BINII) will be performed in cooperation with C6/Zechner and C4/Hermetter. Further, the four predicted Ca-binding sites of APMAP will be mutated to see whether they influence the differentiation capacity of 3T3-L1 cells. Cloning of YFP and CFP constructs will help to discover the exact location of APMAP in the cell and whether addition of Ca2+ is translocating APMAP (microscopy, C10/Kohlwein). To elucidate the possible function in adipogenesis of the other RIKEN genes listed above, their expression levels will be studied in various mouse lines under different conditions (C57/Bl6: regular vs. high fat diet, fasted-fed; OB/OB). In vitro, these genes will be both overexpressed and silenced in 3T3-L1 cells. We will investigate whether their expression levels have an influence on fat cell development, TG hydrolysis/accumulation, LD formation, and on the expression levels of transcription factors known to be important for adipogenesis. In addition, ChIP-qPCR will be performed to find out whether these genes have binding sites for (and might thus be regulated by) transcription factors important in fat cell differentiation. In addition, further candidate genes identified during GOLD III by C2/Trajanoski will be subjected to functional analysis.