A. Interaction of TAF3ΔN with TRF3 is comparable to wildtype TAF3/TRF3 complex. The FLAG-TAF3ΔN/TRF3 complex was purified from insect cells (silver stain). B. TAF3ΔN/TRF3 complex can initiate basal transcription of Myogenin in vitro. Using conditions described in Figure 1C, TAF3ΔN/TRF3 complex was added to our purified transcription system to test the capacity of this complex to initiate transcription of Myogenin. For comparison, an equal amount of TAF3/TRF3 complex was used as a positive control. C. TAF3ΔN/TRF3 complex cannot support activated transcription of Myogenin in vitro. Using similar transcription conditions as in part B, the capacity of TAF3ΔN/TRF3 complex to potentiate activated transcription of Myogenin in the presence of MyoD/E47 was tested. Fold activation was calculated as in Figure 3. Arrowheads indicate transcription product analyzed by primer extension.

A. Representative subunits of CRSP/MED complex are dramatically altered in differentiated myotubes. Cell types derived from C2C12 and fibroblast cells were analyzed by protein immunoblots using various antibodies against CRSP/MED subunits. As a positive control, the same set of protein lysates were also analyzed for TAF3, TRF3 and Myogenin expression. Coomassie staining of the gel loaded with equal amounts of cell lysate was used as loading control. B. Purified CRSP/MED complex is included in reconstituted in vitro transcription of Myogenin. Fold activation is calculated as described in Figure 3. Arrowheads indicate the expected primer extension product.

A. TAF3 directly interacts with MyoD. FLAG-TAF3 and FLAG-TAF3ΔN were expressed in insect cells. Protein lysates were prepared and incubated with glutathione-bound Gst-MyoD or Gst control. Asterisk (*) on Coomassie stain gel indicates degradation band. The samples bound on beads were analyzed by protein immunoblot using anti-FLAG antibody. B. Recovery of Myogenin expression in rescued-TAF3 RNAi cells. C2C12 cells depleted for endogenous TAF3 were grown in both proliferating (P) and differentiation (D) conditions. These cells were then rescued by introducing an RNAi-resistant FLAG-TAF3, FLAG-TAF3ΔN or vector control expression construct. Total RNA was isolated from these TAF3-rescued RNAi cell lines to analyze the recovery of Myogenin expression by quantitative PCR (Q-PCR). Samples were normalized to U6 RNA levels, and represented as mean from four reactions. The error bars represent the standard deviation.

A. Inclusion of purified TBP from insect cells and immunopurified TFIID complex isolated from HeLa cells in our in vitro reconstituted transcription reactions (shown here in silver stain gels). B. TBP and TFIID can initiate basal transcription of Myogenin in vitro. Transcription reactions contain basal transcription factors (TFIIA, B, E, F and H) and RNA Polymerase II. Reactions initiated by purified TRF3 or TAF3/TRF3 were compared to reactions containing TBP or TFIID or TAF3 alone complex to assess the basal level of Myogenin transcription. C. TFIID complex cannot substitute for TAF3/TRF3 function in mediating MyoD- dependent activation of Myogenin. Using conditions described in B, MyoD/E47 were included in reconstituted transcription reactions initiated by TRF3, TAF3/TRF3, TBP or TFIID. As a positive control, a similar amount of TFIID used for Myogenin transcription reaction was used on the TFIID-dependent DNA template G3BCAT in the presence of Sp1. The numerical values represent the fold activation. The fold of MyoD-dependent activation of Myogenin was calculated by dividing the scanned transcriptional signal value generated from reaction done with MyoD over the value obtained from transcription reactions done without the activator. Arrowheads indicate transcription product analyzed by primer extension.

A. Luciferase reporter assay to determine the Myogenin promoter response to the activator MyoD. Diagram of the reporter constructs transfected into 3T3 fibroblast cells. For this reporter assay, pGL3 vector control sequences alone or pGL3 vector containing the endogenous Myogenin promoter sequences (184 bp fragment) were used to drive the expression of luciferase. Constructs were introduced into fibroblast cells in the presence or absence of exogenous FLAG-MyoD. Luciferase expression was determined and plotted as fraction relative to total protein (luciferase/ug protein). Each bar represents the mean of triplicate samples per condition. The error bars represent the standard deviation. Protein immunoblot analyses were performed to determine relative levels of transfected FLAG-MyoD in fibroblast cells. B. TAF3 is required to potentiate MyoD-dependent transcription activation of Myogenin in vitro. Silver stain of the myogenic activator FLAG-MyoD co-expressed with its heterodimer E47 were affinity purified from insect cells using anti-FLAG antibody. This purified factor was included in our purified in vitro reconstituted transcription system to test the effects of activator in TRF3- or TAF3/TRF3-mediated transcription reactions. Using conditions described in Figure 1C, TRF3- or TAF3/TRF3-mediated transcription reactions were supplemented with the purified activator MyoD/E47 in vitro. The arrow indicates specific transcription products analyzed by primer extension.

A. Purified TAF3 interacts with DNA-bound TRF3 in vitro. In electrophoretic mobility shift assay, the labeled 21bp fragment of Myogenin promoter containing the TATA box was used. Lane 1 contains the labelled probe alone; lane 2 contains TRF3 alone while lanes 3–4 include an unlabelled cold TATA competitor (TATA comp.). The binding reaction in lane 5 contains an unlabelled non-specific competitor (TATA mutant, N.S. comp.). The addition of purified TAF3 to reactions containing TRF3 produces a supershifted complex (lane 8 and lane 9 with N.S. comp.). In contrast, in reactions containing TAF3 alone has no DNA-binding activity (lanes 6–7) B. Silver stain of affinity purified basal factors (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH and RNA Polymerase II) and TRF3, TAF3 and TAF3/TRF3 complex used to substitute for TFIID in our in vitro reconstituted transcription reaction. C. Factor requirements for basal transcription of Myogenin mediated by TRF3 or TAF3/TRF3 complex. In vitro transcription reactions contain general transcription factors (TFIIA, B, E, F and H) and RNA Polymerase II. In place of TFIID, purified TRF3 or TAF3/TRF3 complex was substituted to test for transcription initiation properties. In vitro transcription reactions were carried out on Myogenin template containing the endogenous promoter sequence elements (as marked) modified with two additional activator sites (E-boxes) and an upstream CAT gene sequence. Transcription products were analyzed by primer extension using specific primers directed at CAT gene. All transcription reactions unless otherwise indicated contains the Myogenin template, purified basal factors that excludes TFIID. Lane 1 contains DNA template alone while lane 2 only contains purified basal factors. Equal amounts of TRF3, TAF3 and TAF3/TRF3 complex were used in transcription reactions. The arrow indicates specific transcription product.