FIGURE 5.

Tudor-SN combines with the PPRE region and activates downstream gene expression. A, association of Tudor-SN with the PPRE of the aP2 promoter in differentiated 3T3-L1 cells. Cross-linked chromatin from 4-day-differentiated 3T3-L1 cells was incubated with antibodies against PPARγ and Tudor-SN or lgG antibody as a negative control. Primers to the aP2 PPRE region were used for PCR, and primers amplifying a region outside the PPARγ and Tudor-SN binding site were used as a negative control. IP, immunoprecipitate. B, Tudor-SN enhances the transcriptional activity of genes containing a PPRE. 3T3-L1 cells were cotransfected with constructs carrying the PPRE promoter region linked with the Luc reporter gene (0.5 μg) and β-galactosidase vector (0.5 μg) together with Tudor-SN or PPARγ expression plasmids. Normalized luciferase activity is presented. The mean normalized luciferase values of three independent experiments with the S.D. are shown. C, chromatin immunoprecipitation assays demonstrating that knock out of Tudor-SN affects the association of PPARγ with its downstream gene aP2. Cross-linked chromatin from 0-, 5-, and 8-day differentiated MEF-WT or MEF-KO cells were incubated with antibodies against PPARγ or IgG as a negative control, and the primers for the aP2 PPRE region were used for PCR. D, statistical analysis of the ChIP-qPCR results was performed using the independent-samples Student's t test. The results are the means± S.D. *, p < 0.05 (n = 3); ***, p < 0.001(n = 3). E, mRNA expression of aP2 and adipsin in MEF-WT or MEF-KO cells during adipogenesis. Total RNA was harvested from cells treated with DMI at the indicated time points. Real-time PCR analysis was performed to detect the mRNA levels of aP2 and adipsin. Expression levels were normalized to GAPDH mRNA expression.