Interaction of VITO-1 with TEF-1 depends on the SID domain. SDS–gel electrophoresis of in vitro translated, [³⁵S]methionine-labelled TEF-1 (lanes 2), VITO-1-MTAG (lane 3), MTAG-VITO-1 (lane 4), ΔCVITO-1(1–199)-MTAG (lane 5), MTAG-ΔC-VITO-1(1–199) (lane 6), ΔSID-VITO-1(108–323)-MTAG (lane 7), MTAG-ΔSID-VITO-1(108–323) (lane 8), Myf-5 (lane 9) and E2-2 (lane 10). Control immunoprecipitations with an anti-myc epitope antibody (lanes 11–17) and an anti-E2-2 antibody (lane 18). Co-immunoprecipitations of unlabelled VITO-1-MTAG together with labelled TEF-1 (lane 19), unlabelled MTAG-VITO-1 together with labelled TEF-1 (lane 20) and labelled TEF-1 (lanes 21–24) together with labelled ΔCVITO-1(1–199)-MTAG (lane 21), MTAG-ΔCVITO-1(1–199) (lane 22), ΔSID-VITO-1(108–323)-MTAG (lane 23) and MTAG-ΔSID-VITO-1(108–323) (lane 24). A control co-immunprecipation of E2-2- and Myf-5 with an antibody against E2-2 is shown in lane 25. The co-immunoprecipitation clearly demonstrates that VITO-1 and TEF-1 form a complex in the absence of DNA.

TEF-1-mediated transactivation of MCAT-dependent reporter constructs in HEK 293 (A, B, E and F) and C2C12 muscle cells (C and D) depends on VITO-1. HEK 293 cells and C2C12 cells were co-transfected with the reporter construct pTA-LUC-4×MCAT (A–D) and pTA-LUC-1×MCAT (E and F) and pCS2-based expression vectors for TEF-1 and VITO-1, either alone or in combination. The results of three different experimental series each consisting of five independent transfections are shown. Luciferase activities in relative light units (RLU) (A, C and E). Luciferase activities displayed as fold stimulation of pTA-LUC-4×MCAT (A–D) or pTA-LUC-1×MCAT (E and F) background activities. Neither VITO-1 nor TEF-1 alone are sufficient for efficient transactivation of pTA-LUC-4×MCAT or pTA-LUC-1×MCAT while a combination of both leads to an increase in luciferase activity.

(A) VITO-1 is not a direct transcriptional activator. The complete coding region of VITO-1 containing vestigal homology region 1 (VHD-1), vestigal homology region 2 (VHD-2) and the scalloped interaction domain (SID) as well as various parts of the VITO-1 protein were fused to the GAL4 DNA-binding domain (DBD) and analysed on a GAL4 reporter plasmid. Wild-type GAL4 containing the GAL4 transactivation domain (TAD) and a GAL4–VP16 fusion protein were used as positive controls. No significant VITO-1-dependent transactivation activity was detected. (B) VITO-1 does not contribute to MEF2C-mediated activation of MEF2-dependent reporter genes. A functional MEF2 reporter construct (2×MEF2C-TK) and a reporter construct carrying mutated Mef2-binding sites (2×MEF2Cmt-TK) were transfected into HEK293 and C2C12 myoblasts and myotubes either alone (bars 1–3 and 7–9) or together with MEF2C (bars 4–6, 10–12 and 16–21). Addition of VITO-1 either alone (bars 16–18) or in combination with TEF1 (bars 19–21) did not increase activation of the active MEF2 reporter plasmid by MEF2C.

DNA binding of TEF-1 and VITO-1 to different MCAT DNA motifs. Electrophoretic mobility shift assays (EMSA) with in vitro translated TEF-1 (lanes 1–3), TEF-1 and VITO-1 (lanes 4–6), VITO-1 (lanes 7–9) using oligo bs01 (lanes 1, 4, 7 and 10), oligo bs02 (lanes 2, 5 and 8) and oligo bs03 (lanes 3, 6 and 9). Equimolar concentrations of in vitro translated proteins were loaded as estimated from incorporation of ³⁵S per methionine residue present in each protein and densitometric scanning. Between 4 and 8% of the volume of coupled in vitro transcription/translation reaction was used for an EMSA reaction if not indicated otherwise. An unstable, non-specific complex (R) that sometimes formed with bs01 and which was also present in unprimed reticulocyte lysate (lane 10) was clearly distinguishable from TEF-1-dependent complexes (TEF). Addition of VITO-1 led to a reduction in complex formation of TEF-1 with all three binding DNA fragments containing single MCAT motifs (lanes 4–6). No complex formation was noted when VITO-1 was used alone (lanes 7–9). To demonstrate specificity of TEF-1 DNA binding, increasing amounts of unlabelled Bs01 were incubated with the same labelled binding site (lanes 10–15). Lane 11, 0 ng; lane 12, 25 ng (10×); lane 13, 250 ng (100×); lane 14, 500 ng (200×); lane 15, 1250 ng (500×); lane 16, 2500 ng (1000×). FO, free or unbound oligonucleotides.

(A) VITO-1 reduces binding of TEF-1 to single MCAT binding sites. Complex formation of TEF-1 with a single MCAT motif present on bs03 was challenged with increasing concentrations of the bHLH protein Myf-5 (lanes 1–5) and VITO-1 (lanes 6–10) and visualized by EMSA. No reduction in complex formation was discernable (lanes 1–5) after addition of increasing amounts of Myf-5 (lane1, 0%; lane 2, 5%; lane 3, 10%; lane 4, 20%; lane 5, 40% of a translation reaction) while supplementation with the same increasing concentrations of VITO-1 (up to 40% of a translation reaction) led to a clear reduction in TEF-1 binding (lanes 6–11). In a reverse experiment a constant concentration of VITO-1 was titrated against increasing concentrations of TEF-1 (up to 40% of a translation reaction) (lanes 12–15). TEF-1 complex formation was restored when the TEF-1 concentration exceeded the concentration of VITO-1. (B) Binding of TEF-1 to a multimerized MCAT binding site is resistant to modulation by VITO-1. An oligonucleotide containing four copies of the same tandemly repeated MCAT binding motif as used for transfection experiments was analysed for complex formation with TEF-1 (lane 1), VITO-1 (lane 2) and a combination of TEF-1 and VITO-1 (lane 3). Binding assays were primed with 10% of a VITO-1 and 20% of a TEF-1 translation assay to account for differences in the concentration of proteins as judged by densitometric scanning. In contrast to single MCAT binding sites, TEF-1 complexes forming on tandem repeated MCAT motifs were not disrupted by VITO-1. No complex formations on tandem repeated MCAT motifs were observed with TEF-3 under our experimental conditions (lanes 4–6). The unbound oligonucleotides are visible in the lower part (fO).

MyoD-mediated myogenic conversion of C3H10T1/2 cells is modulated by VITO-1 expression levels. C3H10T1/2 cells were transfected with pEMSV-MyoD and pCS2-VITO-1 (A and E), pEMSV-MyoD and pSUPER (B and F), pEMSV-MyoD and pSUPER-VITO-1 (C and G), pEMSV-MyoD and pSUPER-Myogenin (D and H) and stained after 72 h with an antibody against Myogenin (A–D) or MyHC (E–H). Cumulative results of myogenic conversion experiments (I). The number of myogenic cells obtained after transfection of MyoD was set as 100%. Supplementary expression of VITO-1 led to an improvement in MyoD-mediated myogenic conversion while knockdown of VITO-1 or Myogenin expression decreases muscle cell formation. Transfection efficiencies were normalized against a co-transfected LacZ expression construct.

Knockdown of VITO-1 expression attenuates differentiation of C2C12 muscle cells. C2C12 myoblasts were co-transfected with pSUPER and pEGFP-C2 (A and E), pSUPER-Vito-1 and pEGFP-C2 (B, C, F and G) and pSUPER-Myogenin and pEGFP-C2 (D and H). Cells were examined for EGFP fluorescence to visualize transfected cells (E–H) and stained with a Myogenin antibody (G) (detected with a rhodamine coupled secondary antibody) or inspected by phase contrast microscopy (A–D). Note that transfected cells expressing VITO-1 siRNA (green) are negative for Myogenin (red). Knockdown of VITO-1 and Myogenin expression led to an inhibition of myotube formation while differentiation of C2C12 cells occurred normally after transfection with pSUPER.