P300/CBP acts as a coactivator to cartilage homeoprotein-1 (Cart1), paired-like homeoprotein, through acetylation of the conserved lysine residue adjacent to the homeodomain.
The paired-like homeoprotein, Cart1, is involved in skeletal development. We describe here that the general coactivator p300/CBP controls the transcription activity of Cart1 through acetylation of a lysine residue that is highly conserved in other homeoproteins. Acetylation of this residue increases the interaction between p300/CBP and Cart1 and enhances its transcriptional activation.


BACKGROUND
Cart1 encodes a paired-like homeoprotein expressed selectively in chondrocyte lineage during embryonic development. Although its target gene remains unknown, gene disruption studies have revealed that Cart1 plays an important role for craniofacial bone formation as well as limb development by cooperating with another homeoprotein, Alx4. In this report, we study the functional involvement of p300/CBP, coactivators with intrinsic histone acetyltransferase (HAT) activity, in the transcriptional control of Cart1.


METHODS
To study the transcription activity of Cart1, a reporter construct containing a putative Cart1 binding site was transiently transfected with the expression vectors of each protein. The interaction between p300/CBP and Cart1 was investigated by glutathione S-transferase (GST) pull-down, yeast two-hybrid, and immunoprecipitation assays. In vitro acetylation assay was performed with the recombinant p300-HAT domain and Cart1 in the presence of acetyl-CoA.


CONCLUSIONS
p300 and CBP stimulate Cart1-dependent transcription activity, and this transactivation is inhibited by E1A and Tax, oncoproteins that suppress the activity of p300/CBP. Cart1 binds to p300 in vivo and in vitro, and this requires the homeodomain of Cart 1 and N-terminal 139 amino acids of p300. Confocal microscopy analysis shows that Cart1 recruits overexpressed and endogenous p300 to a Cart1-specific subnuclear compartment. Cart1 is acetylated in vivo and sodium butyrate and trichostatin A, histone deacetylase inhibitors, markedly enhance the transcription activity of Cart1. Deletion and mutagenesis analysis identifies the 131st lysine that locates immediately adjacent to the homeodomain as a target of p300-HAT, and a point mutation to this residue attenuates the binding affinity to p300 as well as p300-dependent transcription activity. Together, these results indicate that p300/CBP acts as a cotransactivator to Cart1 through a direct interaction and specific lysine acetylation. In addition, because 131st lysine is highly conserved in other types of homeoprotein, this lysine may be a common target for HAT of p300/CBP for these proteins.
Iioka
Takashi
T
Division of Orthopaedic Pathomechanism, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Furukawa
Keizo
K
Yamaguchi
Akira
A
Shindo
Hiroyuki
H
Yamashita
Shunichi
S
Tsukazaki
Tomoo
T
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050881:E1A-Associated p300 Protein; D018398:Homeodomain Proteins; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
C489985:ALX1 protein, human; C489986:Alx1 protein, mouse; C083282:Alx1 protein, rat; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse; C494362:Ep300 protein, rat; D008239:Lysine