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. 2008 May 16;283(20):13611–13626. doi: 10.1074/jbc.M800128200

FIGURE 1.

FIGURE 1.

In vitro studies. A, scheme of the rat glut4 upstream region. The rat glut4 promoter region (1 kb) contains the three CpG islands, MyoD-I, MyoD-II, and the MEF2 DNA binding consensus elements 5′ to the transcriptional (+1) and translational (ATG) start sites. In addition, NF-1, Sp-1, PU.1, and WT-1 consensus sequences are shown. The sequences deleted in mutant (Δ) constructs are depicted in parentheses. B, transient transfection of glut4-luciferase DNA constructs in C2C12 murine skeletal muscle cell line. Luciferase reporter activity of wild type (WT) and mutated glut4-luciferase DNA constructs is depicted as a ratio to that of the β-galactosidase gene. Differences between the wild type and various mutated constructs were established by analysis of variance (F = 11.6; p = 0.0001), and inter-group differences in comparison with the WT DNA construct were determined by the post-hoc Tukey's HSD test (* and **). Mutant CpG-II and MyoD-II activate (Ac) and MEF2 de-activates (de-Ac) luciferase gene transcription. C, DNA methylation. glut4-luciferase DNA construct was either pretreated or untreated with the SssI methylase enzyme. Top panel, demonstrates the methylated and unmethylated glut4 DNA that was restricted with methylation-sensitive HpaII and BstU1 enzymes, and the digested DNA products were separated by gel electrophoresis along with the unrestricted DNA. Although the unmethylated glut4 DNA was restricted yielding smaller products, the SssI methylase-treated methylated DNA was resistant to restriction enzyme digestion yielding larger DNA products. M = DNA markers. Lower panel demonstrates the luciferase activity of SssI methylase-treated and untreated glut4-luciferase (0–4 μg) transiently transfected in C2C12 cells. Differences between DNA concentration-matched methylated and unmethylated DNA-reporter activity was assessed by the Student's t test (*). D, histone de-acetylation. Transiently transfected C2C12 cells containing wild type (WT) and mutated glut4-luciferase DNA constructs were pretreated with trichostatin A (0.6 μm), a generic HDAC inhibitor. The mutant constructs were compared with the WT construct by analysis of variance (F = 96.1; p = 0.0001), and inter-group differences were established by the post-hoc Tukey's HSD test (*, **). E, in vitro nutrient restriction. The transiently transfected cells containing the wild type glut4-luciferase DNA construct were incubated in either a control or a nutrient-restricted (glucose and amino acid deprived) medium. Luciferase activity is depicted as a ratio to the β-galactosidase gene (internal control) per protein concentration. Student's t test demonstrated inter-group differences (*).