Functional mapping of the MH1 DNA-binding domain of DPC4/SMAD4

The transcription factor Smad4 binds DNA in response to a TGF-beta ligand-i nitiated intracellular signaling cascade. SMAD4 is deleted or mutated durin g tumorigenesis in many human tumors. Some of these mutations occur in the N-terminal portion of the protein, the Mad homology 1 (MH1) region, which e xhibits sequence-specific DNA-binding. We used alanine scanning mutagenesis and natural mutations to map the subregion of the MH1 domain necessary for that function. We created 20 individual mutations in the MH1 region of hum an Smad4 and assayed their effect on DNA-binding in vitro. Mutation of resi dues in the less conserved N- and C-terminal areas of the MH1 region had no effect on DNA-binding. However, mutations in the domain from L43 to R135 c aused a dramatic reduction of the ability of Smad4 to bind DNA. Previous wo rk demonstrated a beta-hairpin protein motif within this region to be respo nsible for DNA-binding, but suggested that the tumorigenic mutations occurr ing outside this motif may target a separate function of the MH1 domain. Ou r results demonstrate that the MH1 domain as a whole is very sensitive to c hanges in overall structure, and that tumorigenic mutations within the regi on of L43-R135 indeed would target DNA-binding.