Functional domains of paired-like homeoprotein Cart1 and the relationship between dimerization and transcription activity.
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.


CONCLUSIONS
These results revealed that homodimerization through direct interaction is necessary for the potent transcription activity of Cart1.
Furukawa
Keizo
K
Department of Orthopaedic Surgery, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Nagasaki 852-8523, Japan.
Iioka
Takashi
T
Morishita
Mariko
M
Yamaguchi
Akira
A
Shindo
Hiroyuki
H
Namba
Hiroyuki
H
Yamashita
Shunichi
S
Tsukazaki
Tomoo
T
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015139:Blotting, Southern; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D019281:Dimerization; D018398:Homeodomain Proteins; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation
C083282:Alx1 protein, rat; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D020033:Protein Isoforms