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. 2006 Feb 24;394(Pt 3):699–706. doi: 10.1042/BJ20051303

Figure 5. TauT mRNA expression is transcriptionally regulated by MEF2C during skeletal muscle differentiation in C2C12 cells.

Figure 5

(A) Sequence alignment of the −858/−813 rat TauT promoter and the equivalent regions in the human and mouse TauT genes. The MEF2 consensus region is underlined. (B) MEF2 binds directly to the MEF2 consensus sequence (−844/−835) of the TauT promoter. C2C12 myoblasts were differentiated into myotubes by culturing in DM for 2 days. Nuclear extracts were prepared from C2C12 myoblasts or myotubes. The probe containing the MEF2 consensus sequence was labelled and incubated with nuclear extracts. The formation of DNA–protein complexes was analysed by EMSAs in the presence or absence of 100-fold excess of unlabelled probes with (mut) or without (wt) mutation at MEF2 site. The supershift assay was carried out using anti-MEF2 antibody. (C) The MEF2-binding site at −844 to −835 functionally regulates TauT transcriptional activation in myotubes. C2C12 cells were transfected with promoter–reporter constructs (pTauT/−862-luc or pTauT/−862 mut-luc or pTauT/−834-luc or pl-luc) and pRL-TK and cultured in GM for 18 h. Then cells were cultured in GM or DM for 2 days and then harvested. The basic vector, pl-luc, was used as a promoterless control. The promoter activity of the TauT gene was measured and normalized against the luciferase activity of pRL-TK. Assays were performed in triplicate. Results are means±S.E.M., n=3. The experiments were repeated three times with similar results. *, P<0.05 and **, P<0.01 compared with GM; #, P<0.05 and ##, P<0.01 compared with DM pTauT/−862-luc. SS, supershifted band.