HIGH-AFFINITY BINDING OF MEF-2C CORRELATES WITH DNA BENDING

To regulate lineage-specific gene expression in many. cell types, memb ers of the myocyte enhancer factor-2 (MEF-2) family of transcription f actors cooperate with basic helix-loop-helix (bHLH) proteins, which sh ow only limited intrinsic DNA binding specificity, We investigated the DNA binding properties of MEF-2C in vitro and show that the inherent bendability of the MEF site is one of the principal structural charact eristics recognized by MEF-2C, Measurements of the apparent dissociati on constants of MEF-2C complexes with several DNA sequences revealed t hat MEF-2C bound with high affinity to DNA sequences containing a MEF site, Mutations in the MEF site which did not affect the bendability o f the DNA changed the free energy of binding only marginally, However, reducing the intrinsic bendability of the DNA binding site through an AA --> GC substitution increased the half-maximal binding concentrati on of MEF-2C by almost one order of magnitude, Electrophoretic mobilit y shift assays revealed markedly reduced MEF-2C binding to DNA contain ing 2, 6-diaminopurine. On binding to MEF-2C the maximum ellipticity a t 275 nm in the CD spectrum of DNA containing a MEF site was red shift ed by 4 nm and its intensity reduced significantly, white a slight blu e shift of < 1 nm was observed for a mutant DNA sequence with reduced bendability (AA --> GC). Bending analysis by circular permutation assa y revealed that the DNA in the cognate complex was bent by 49 degrees, while the DNA in the complex with the mutant oligonucleotide was larg ely unbent.