Cos-7 cells were transiently transfected with vectors containing human PPARG1 (provided by V.K.
Cos7 cells were transfected with human PPARG1 and RXRA expression vectors and a PPRE-driven reporter construct and treated with vehicle or FM550.
(A) Cos-7 cells were transiently transfected with human PPARG1 and PPRE x3-TK-luc, with either pcDNA3 or PPAR[gamma]-DN vectors.
In this study, association of DNA methylation levels and PPARG1 and FABP4 gene expression levels were examined in the IMF and muscle portions of the longissimus dorsi muscle (LM) tissues in Korean cattle steers.
To detect expression levels of PPARG1 and FABP4 genes, total RNA was isolated as previously described (Jeong et al., 2013) using TRIzol reagent (Molecular Research Center, Cincinnati, OH, USA).
To determine DNA methylation levels, target regions of PPARG1 (Figure 1a) and FABP4 (Figure 2a) genes were selected from the CpG islands, which were searched using CpG Island Searcher (USC Norris Comprehensive Cancer Center, USA; http://www.uscnorris.com/cpgislands2/cpg.aspx).
Real-time PCR analysis showed that mRNA levels of both PPARG1 (Figure 1b; p<0.01) and FABP4 (Figure 2b; p<0.001) genes were higher in the IMF portion than in the muscle portion of the LM.
Next, we determined DNA methylation levels within CpG island promoter regions of the PPARG1 and FABP4 genes.
Indeed, the human PPARG gene consists of nine exons and--by differential promoter's usage and alternative splicing--generates at least four main splice variants (i.e., PPARG1, PPARG2, PPARG3, and PPARG4).
Using specific primers pairs (Table 1), we performed an extensive expression analysis of PPARG1, PPARG2, PPARG3, PPARG4, and ORF4 in tissues and cells related to complications of metabolic syndrome--such as altered glucose and lipids' metabolism (liver), increased inflammatory response (macrophages), atherosclerosis (EPCs, heart, and macrophages), cancer (colon carcinoma and MCF7), and thyroid dysfunction (thyroid)--and in a widely used cell model, HEK293 [3, 6, 17, 39, 40].
The tissue-specific expression pattern of PPARG alternative variants, including also transcripts encoding the same protein (PPARG1, PPARG3, and PPARG4), is shown in Figure 2.
This analysis revealed that PPARG1 and PPARG4 are the only canonical transcripts contributing to the final expression of PPAR[gamma] protein in undifferentiated hMSCs and therefore that these cells express only PPAR[gamma]1 isoform.