FIGURE 6.

MyoD and SREBP-1 regulate ANKRD2 expression. A, schematic representation of Ankrd2 promoter. The region between −1700 and +1 bp contains putative binding elements for MyoD (green), NF-κB (red), and SREBP-1 (pink). B, chromatin was isolated from the skeletal muscles of WT and mdm mice and precipitated with anti-c-MyoD, anti-NF-κB, anti-SREBP-1, anti-RNA poly II, or nonspecific IgG. qPCRs were performed with three sets of primers, specific for ANKRD2 promoter to identify the specific transcription factor and its region of binding to the ANKRD2 promoter and resolved in 1% agarose gel. C, 1500-bp pGL-WT, 700-bp (pGL-mtMyoD), or 600-bp (pGL-SREBP-1) promoter regions were synthesized and linked to luciferase (Luc) reporter gene. D, pMB^WT and pMB^mdm were transfected with empty vector, pGL-WT, pGL-mtMyoD, or pGL-SREBP-1 vector. Forty-eight h after transfection, firefly luciferase activities were estimated and normalized to Renilla luciferase activities. E and F, MB^WT were transfected with MyoD siRNA or nonspecific siRNA (NS siRNA) followed by transfection of either pGL or pGL-WT. MB^mdm were transfected with SREBP-1 siRNA or nonspecific siRNA followed by transfection of either pGL or pGL-WT. After 48 h, total protein was isolated, and MyoD and SREBP-1 protein expression was determined by Western blot (E). Forty-eight h after transfection, firefly luciferase activities were estimated and normalized to Renilla luciferase activities (F). Gel pictures are representative of three separate experiments. Each error bar indicates mean ± S.E. (n = 3). *, p < 0.05.