Csx/Nkx2.5 is an evolutionarily conserved homeodomain (HD)-containing transcription factor that is essential for early cardiac development. We found that the HD of Csx/Nkx2.5 binds as a monomer as well as a dimer to its DNA binding sites in the promoter of the atrial natriuretic factor (ANF) gene, an in vivo target gene of Csx/Nkx2.5. Csx/Nkx2.5 physically interacts with each other in vitro as well as in cells, and the HD is critical for homodimerization. Lys 193 and Arg 194 , located at the

more »

... H-terminal end of HD, are essential for dimerization. Lys 193 is also required for a specific interaction with the zinc finger transcription factor GATA4. Csx/ Nkx2.5 can heterodimerize with other NK2 homeodomain proteins, Nkx2.3 and Nkx2.6/Tix, with different affinities. A single missense mutation, Ile 183 to Pro in the HD of Csx/Nkx2.5, preserved homodimerization function, but totally abolished DNA binding. Ile 183 3 Pro mutant acts in an inhibitory manner on wild type Csx/ Nkx2.5 transcriptional activity through the ANF promoter in 10T1/2 cells. However, Ile 183 3 Pro mutant does not inhibit wild type Csx/Nkx2.5 function on the ANF promoter in cultured neonatal cardiac myocytes, possibly due to failure of dimerization in the presence of the target DNA. These results suggest that complex proteinprotein interactions of Csx/Nkx2.5 play a role in its transcriptional regulatory function. GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MSV, murine sarcoma virus.