THE N-TERMINAL METHIONINE IS A MAJOR DETERMINANT OF THE DNA-BINDING SPECIFICITY OF MEF-2C

Members of the MEF-S family of transcriptional regulators positively m odulate the activity of basic helix-loop-helix proteins in both myogen ic and neurogenic cell lineages. Previous work had shown that MEF-2C(2 -117), a protein fragment comprising the dimerization and DNA-binding domains of MEF-2C but lacking the N-terminal methionine, bound to AT-r ich DNA sequences with high affinity. MEF-2C(2-117) did not discrimina te between different AT-rich sequences. We now report the in vitro DNA binding properties of a MEF-2C fragment containing the N-terminal met hionine. Measurements of the apparent dissociation constants of the co mplexes of GG-MEF-2C(1-117) revealed that different AT-rich sequences are bound with different affinities; in particular MEF site containing DNA (CTATAAATAG) is bound preferentially to DNA containing a SRF site (CATAAATG). Strikingly, when the shorter AT run consisted of six alte rnating thymines and adenines, almost wild-type affinity was observed. Irrespective of the particular DNA sequence, all circular dichroism s pectra of the DNA complexes of GG-MEF-2C(1-117) were superimposable an d characterized by an identical maximal ellipticity at 269.5 nm, sugge sting similar DNA conformations. Bending analysis by circular permutat ion assay revealed that on complex formation MEF-2C(2-117) induced cog nate DNA to bend by 49 degrees, while heterologous DNA remained unbent . In the presence of the N-terminal methionine, however, all DNA seque nces were bent by 70 degrees. The above results suggest an important f unction for the N-terminal methionine in properly orientating MEF-2C o n the DNA.