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. 2013 Apr;33(7):1368–1382. doi: 10.1128/MCB.01259-12

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Fig 4 — ZEB1 binds to G/C-centered E-boxes in muscle differentiation genes whose transcription is repressed largely through a CtBP-dependent mechanism. (A) ZEB1 and MyoD display differential binding to G/C-centered E-boxes in muscle differentiation genes in myoblasts and myotubes. Shown are data for qRT-PCR of fragments of the mouse Myh4, troponin T1, and GAPDH promoters immunoprecipitated in ChIP assays from C2C12 myoblasts and myotubes with ZEB1 antibody (E-20X), MyoD antibody (C-20), or their respective IgG controls. ChIP assays were performed as described in Materials and Methods. Values represent binding relative to the input averaged from two experiments, each performed in triplicate, and are representative of at least three essays. (B) The basal transcriptional activity of the MHC promoter is under negative regulation by ZEB1 and CtBP in myoblasts but not in myotubes. Nonconfluent cycling C2C12 myoblasts or confluent myotubes allowed to terminally differentiate for 48 h were cotransfected with 100 nM control siRNA (siCtl) or siRNAs against ZEB1 (si1ZEB1 and si2ZEB1) or CtBP (siCtBP) or equal molar amounts of their different combinations along with 0.4 μg of a luciferase reporter for the MHC promoter. Transcriptional assays and assessment of relative luciferase units (RLU) in panels B to E, G, and H were performed as described in Materials and Methods and are representative of at least four independent experiments. The knockdown efficiencies of si1ZEB1, si2ZEB1, and siCtBP in C2C12 cells are shown in panel F and in Fig. 1A to C. (C) Same as panel B but with 0.2 μg of the troponin I promoter as a luciferase reporter. (D) Same as panel B but with 0.2 μg of the MCK promoter as a luciferase reporter. (E) Same as panel B but with 0.2 μg of the MyoD enhancer/promoter. (F) Knockdown efficiency of siRNAs against CtBP and ZEB1 in C2C12 and C3H-10T1/2 cells. (Left) C2C12 cells were transfected with 100 nM either siCtl or siCtBP. CtBP protein levels were assessed by Western blotting using an antibody against CtBP1/2 (E-12) along with α-tubulin (B5-1-2) as a loading control. (Middle) C3H-10T1/2 cells were transfected with 200 nM either siCtl or si1ZEB1. ZEB1 protein levels were assessed by Western blotting using an antibody against ZEB1 (H-102) along with α-tubulin (B5-1-2) as a loading control. (Right) Same as the left panel but with C3H-10T1/2 cells. (G) Muscle genes are also under negative regulation by ZEB1 and CtBP in the C3H-10T1/2 myogenic conversion model. C3H-10T1/2 fibroblasts were cotransfected with luciferase reporters for muscle gene promoters, as in panels B to E, plus either 100 nM siRNA control (siCtl) or specific siRNAs against ZEB1 (siZEB1) or CtBP1/2 (siCtBP). The knockdown efficiencies of si1ZEB1 and siCtBP in C3H-10T1/2 fibroblasts are shown in panel F. (H) Transcriptional repression of muscle genes by ZEB1 depends on CtBP. C3H-10T1/2 fibroblasts were cotransfected with luciferase reporters for muscle gene promoters as in panel G, 0.6 μg of an expression vector for MyoD, and combinations of either 0.4 μg of an expression vector for ZEB1 or equal molar amounts of its corresponding empty expression vectors plus either 100 nM siRNA control (siCtl) or a specific siRNA against CtBP1/2 (siCtBP).

Fig 4 — ZEB1 binds to G/C-centered E-boxes in muscle differentiation genes whose transcription is repressed largely through a CtBP-dependent mechanism. (A) ZEB1 and MyoD display differential binding to G/C-centered E-boxes in muscle differentiation genes in myoblasts and myotubes. Shown are data for qRT-PCR of fragments of the mouse Myh4, troponin T1, and GAPDH promoters immunoprecipitated in ChIP assays from C2C12 myoblasts and myotubes with ZEB1 antibody (E-20X), MyoD antibody (C-20), or their respective IgG controls. ChIP assays were performed as described in Materials and Methods. Values represent binding relative to the input averaged from two experiments, each performed in triplicate, and are representative of at least three essays. (B) The basal transcriptional activity of the MHC promoter is under negative regulation by ZEB1 and CtBP in myoblasts but not in myotubes. Nonconfluent cycling C2C12 myoblasts or confluent myotubes allowed to terminally differentiate for 48 h were cotransfected with 100 nM control siRNA (siCtl) or siRNAs against ZEB1 (si1ZEB1 and si2ZEB1) or CtBP (siCtBP) or equal molar amounts of their different combinations along with 0.4 μg of a luciferase reporter for the MHC promoter. Transcriptional assays and assessment of relative luciferase units (RLU) in panels B to E, G, and H were performed as described in Materials and Methods and are representative of at least four independent experiments. The knockdown efficiencies of si1ZEB1, si2ZEB1, and siCtBP in C2C12 cells are shown in panel F and in Fig. 1A to C. (C) Same as panel B but with 0.2 μg of the troponin I promoter as a luciferase reporter. (D) Same as panel B but with 0.2 μg of the MCK promoter as a luciferase reporter. (E) Same as panel B but with 0.2 μg of the MyoD enhancer/promoter. (F) Knockdown efficiency of siRNAs against CtBP and ZEB1 in C2C12 and C3H-10T1/2 cells. (Left) C2C12 cells were transfected with 100 nM either siCtl or siCtBP. CtBP protein levels were assessed by Western blotting using an antibody against CtBP1/2 (E-12) along with α-tubulin (B5-1-2) as a loading control. (Middle) C3H-10T1/2 cells were transfected with 200 nM either siCtl or si1ZEB1. ZEB1 protein levels were assessed by Western blotting using an antibody against ZEB1 (H-102) along with α-tubulin (B5-1-2) as a loading control. (Right) Same as the left panel but with C3H-10T1/2 cells. (G) Muscle genes are also under negative regulation by ZEB1 and CtBP in the C3H-10T1/2 myogenic conversion model. C3H-10T1/2 fibroblasts were cotransfected with luciferase reporters for muscle gene promoters, as in panels B to E, plus either 100 nM siRNA control (siCtl) or specific siRNAs against ZEB1 (siZEB1) or CtBP1/2 (siCtBP). The knockdown efficiencies of si1ZEB1 and siCtBP in C3H-10T1/2 fibroblasts are shown in panel F. (H) Transcriptional repression of muscle genes by ZEB1 depends on CtBP. C3H-10T1/2 fibroblasts were cotransfected with luciferase reporters for muscle gene promoters as in panel G, 0.6 μg of an expression vector for MyoD, and combinations of either 0.4 μg of an expression vector for ZEB1 or equal molar amounts of its corresponding empty expression vectors plus either 100 nM siRNA control (siCtl) or a specific siRNA against CtBP1/2 (siCtBP).