Functional domains of the LIM homeodomain protein Xlim-1 involved in negative regulation, transactivation, and axis formation in Xenopus embryos
Functional domains of the LIM homeodomain protein Xlim-1 involved in negative regulation, transactivation, and axis formation in Xenopus embryos
复制标题
DOI：
10.1006/dbio.2000.9986
复制
发表时间：
2001-01-15
影响因子：
2.7
通讯作者：
Taira, M
中科院分区：
文献类型：
--
作者：
Hiratani, I;Mochizuki, T;Taira, M
非洲爪蟾LIM同源结构域蛋白Xlim-1在Spemann组织者区特异性表达，并被认为在体轴的建立中作为转录激活因子发挥作用。为了进一步阐明Xlim-1转录活性调控的机制，我们将Xlim-1同源结构域（CT 239 -403）的C端区域分为5个区域，CCR 1 -5（C端保守区），这是基于Xlim-1与其同源结构域Xlim-5之间的相似性。Xlim-1 CT 239 -403在Spemann组织者中的作用通过测定一系列CCR突变的构建体在非洲爪蟾胚胎中的轴形成能力来分析。我们发现，高剂量的Xlim-1结构删除的CCR 1或CCR 2启动二级轴形成的情况下，其辅激活因子Ldb 1（LIM-domain-binding protein 1），表明CCR 1和CCR 2参与Xlim-1的负调控。相比之下，虽然Xlim-1能够在Ldb 1存在下以低剂量启动二级轴形成，但CCR 2（aa 275-295）的缺失或CCR 2中的五个保守酪氨酸被丙氨酸取代（CCR 2 - 5 YA）消除了活性。此外，非洲爪蟾中的UAS-GAL 4单杂交报告基因分析表明，CCR 2，而不是CCR 2 - 5 YA，其南京区域（aa 261-315）作为一个反式激活结构域时，融合到GAL 4 DNA结合结构域。最后，我们发现，没有已知的转录辅激活因子（CBP，SRC-1，和TIF 2）与Xlim-1的反式激活结构域（aa 261-315）相互作用。因此，Xlim-1不仅包含一个独特的酪氨酸丰富的激活结构域，但也包含一个负调控结构域在CT 239 -403，这表明了一个复杂的调控机制的转录活性的Xlim-1的组织者。(C)北京大学出版社.
Xenopus LIM homeodomain protein Xlim-1 is specifically expressed hi the Spemann organizer region and assumed to play a role in the establishment of the body axis as a transcriptional activator. To further elucidate the mechanism underlying the regulation of its transcriptional activity, we focused on the region C-terminal to the homeodomain of Xlim-1 (CT239-403) and divided it into five regions, CCR1-5 (C-terminal conserved regions), based on similarity between Xlim-1 and its paralog, Xlim-5. The role of Xlim-1 CT239-403 in the Spemann organizer was analyzed by assaying the axis-forming ability of a series of CCR-mutated constructs in Xenopus embryos. We show that high doses of Xlim-1 constructs deleted of CCR1 or CCR2 initiate secondary axis formation in the absence of its coactivator Ldb1 (LIM-domain-binding protein 1), suggesting that CCR1 and CCR2 are involved in negative regulation of Xlim-1. In contrast, while Xlim-l is capable of initiating secondary axis formation at low doses in the presence of Ldb1, deletion of CCR2 (aa 275-295) or substitution of five conserved tyrosines in CCR2 with alanines (CCR2-5YA) abolished the activity. In addition, UAS-GAL4 one-hybrid reporter assays in Xenopus showed that CCR2, but not CCR2-5YA, with its nanking regions (aa 261-315) functions as a transactivation domain when fused to the GAL4 DNA-binding domain. Finally, we show that none of the known transcriptional coactivators tested (CBP, SRC-I, and TIF2) interacts with the Xlim-l transactivation domain (aa 261-315) Thus, Xlim-l not only contains a unique tyrosine-rich activation domain but also contains a negative regulatory domain in CT239-403, suggesting a complex regulatory mechanism underlying the transcriptional activity of Xlim-l in the organizer. (C) 2000 Academic Press.