Figure 4.

MLX and MAX/MDX1 are recruited to Tug1 promoter to coordinate Tug1 repression. A, MLX but not its dominant-negative mutant (DN-MLX) can repress the transcription of Tug1. Cultured mouse podocytes were transfected with Tug1-1kb promoter luciferase construct, together with the indicated Mlx overexpression plasmids or empty vector (Vec) under normal glucose conditions (n = 3). Luciferase readings are presented as mean ± S.E.M. **, p < 0.01; ***, p < 0.001 (compared with vector group); n.s., not significant. B, sequence of glucose-responsive ChoRE and its neighboring E-box in the promoter of mouse Tug1 in WT, ChoRE mutant, E-box mutant, and E-box deletion mutant (ΔE-box). C, both ChoRE (green bar) and E-box (orange bar) bring repressive transcription factors to downregulate Tug1 transcription. Cultured mouse podocytes were transfected with the indicated luciferase constructs of Tug1-1kb promoter (WT), ChoRE and E-box mutants, or empty pGL4 vector (Vec) under normal glucose conditions. Luciferase assays were performed as in A (n = 3). D, MAX and MXD1 can repress Tug1 transcription. Cultured mouse podocytes were transfected with the Tug1-1kb promoter luciferase construct (Tug1-1kb), together with plasmids for the overexpression of the indicated transcription factors or empty pRK5 vector (Vector) under normal glucose conditions. Luciferase assays were performed as in A (n = 3). E, ChIP-qPCR analysis of Tug1 promoter region by the indicated antibodies in podocytes cultured under NG or HG conditions (n = 3). Data are presented as mean ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant (compared with NG group). F, E-box is required for HDAC1-mediated transcription repression of Tug1. Cultured mouse podocytes were transfected with the indicated Tug1-1kb promoter luciferase constructs (WT or E-box mutant) and treated with the indicated concentrations of the HDAC inhibitor TSA or vehicle DMSO for 24 h under normal glucose conditions. Luciferase assays were performed as in A (n = 3).