Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

CSX/NKX2.5 is an evolutionarily conserved homeodomain-containing (HD-contai ning) transcription factor that is essential for early cardiac development. Recently, ten different heterozygous CSX/NKX2.5 mutations were found in pa tients with congenital heart defects that are transmitted in an autosomal d ominant fashion. To determine the consequence of these mutations, we analyz ed nuclear localization, DNA binding, transcriptional activation, and dimer ization of mutant CSX/NKX2.5 proteins. All mutant proteins were translated and local:ed to the nucleus, except one splice-donor site mutant whose prot ein did not accumulate in the cell. All mutants that had truncation or miss ense mutations in the HD had severely reduced DNA binding activity and litt le or no transcriptional activation function. In contrast, mutants with int act HDs exhibit normal DNA binding to the monomeric binding site but had th ree- to ninefold reduction ill DNA binding to the dimeric binding sites. HD missense mutations that preserved homodimerization ability inhibited the a ctivation of atrial natriuretic factor by wild-type CSX/NKX2.5. Although ou r studies do not characterize the genotype-phenotype relationship of the te n human mutations, they identify specific abnormalities of CSX/NKX2.5 funct ion essential for transactivation of target genes.