Iioka Takashi, Furukawa Keizo, Yamaguchi Akira, Shindo Hiroyuki, Yamashita Shunichi, Tsukazaki Tomoo
Division of Orthopaedic Pathomechanism, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
J Bone Miner Res. 2003 Aug;18(8):1419-29. doi: 10.1359/jbmr.2003.18.8.1419.
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.
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.
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.
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.
配对样同源结构域蛋白Cart1参与骨骼发育。我们在此描述，通用共激活因子p300/CBP通过对一个在其他同源结构域蛋白中高度保守的赖氨酸残基进行乙酰化作用来控制Cart1的转录活性。该残基的乙酰化增加了p300/CBP与Cart1之间的相互作用，并增强其转录激活作用。
Cart1编码一种在胚胎发育期间在软骨细胞谱系中选择性表达的配对样同源结构域蛋白。尽管其靶基因尚不清楚，但基因敲除研究表明，Cart1通过与另一种同源结构域蛋白Alx4协同作用，在颅面骨形成以及肢体发育中发挥重要作用。在本报告中，我们研究了具有内在组蛋白乙酰转移酶（HAT）活性的共激活因子p300/CBP在Cart1转录调控中的功能作用。
为研究Cart1的转录活性，将含有假定的Cart1结合位点的报告基因构建体与每种蛋白质的表达载体一起瞬时转染。通过谷胱甘肽S-转移酶（GST）下拉、酵母双杂交和免疫沉淀试验研究p300/CBP与Cart1之间的相互作用。在乙酰辅酶A存在的情况下，用重组p300-HAT结构域和Cart1进行体外乙酰化试验。
p300和CBP刺激依赖Cart1的转录活性，并且这种反式激活被E1A和Tax抑制，E1A和Tax是抑制p300/CBP活性的癌蛋白。Cart1在体内和体外均与p300结合，这需要Cart1的同源结构域和p300的N端139个氨基酸。共聚焦显微镜分析表明，Cart1将过表达的和内源性的p300募集到一个Cart1特异性的核内亚区室。Cart1在体内被乙酰化，组蛋白去乙酰化酶抑制剂丁酸钠和曲古抑菌素A显著增强Cart1的转录活性。缺失和诱变分析确定紧邻同源结构域的第131位赖氨酸是p300-HAT的作用靶点，对该残基的点突变减弱了与p300的结合亲和力以及p300依赖的转录活性。总之，这些结果表明p300/CBP通过直接相互作用和特异性赖氨酸乙酰化作用作为Cart1的共转录激活因子。此外，由于第131位赖氨酸在其他类型的同源结构域蛋白中高度保守，该赖氨酸可能是p300/CBP对这些蛋白进行HAT作用的共同靶点。
