Background: Cart1 encodes the paired-like homeodomain in the central portion of the gene, and plays a crucial role in the developmental lineage of bone and cartilage, especially in head formation. However, its transactivation mechanism is still poorly understood, including the target gene. Here, we report biochemical dissections of Cart1 functional domains and a relationship between dimerization and transcription activity.
Results: Deletion studies of GAL4-fused Cart1 indicated that the transactivation domain is located in the middle portion of the C-terminal domain, but the N-terminal is also required for a full activation of the consensus palindromic binding site (TAATNNNATTA). Analysis of the basic amino acid residues at both ends of the homeodomain revealed that both sides act as nuclear localization signals, and are necessary for the cooperative binding to the palindromic sequence. In this study, two additional Cart1 isoforms that behave as dominant negatives were identified from rat chondrosarcoma cells. These isoforms suppressed the transcription activity of the wild-type, despite loss of DNA binding ability, and could interact with the wild-type in yeast. Finally, we demonstrated that wild-type Cart1 forms a DNA-independent homodimer in in vivo conditions, and that the transactivation of wild-type Cart1 was suppressed by the N- or C-terminal domain which was expressed in the nucleus.
Conclusion: These results revealed that homodimerization through direct interaction is necessary for the potent transcription activity of Cart1.