The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression
The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression
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DOI：
10.1128/mcb.18.6.3120
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发表时间：
1998-06-01
影响因子：
5.3
通讯作者：
Izumo, S
中科院分区：
文献类型：
--
作者：
Lee, Y;Shioi, T;Izumo, S
心肌谱系的特化和分化似乎需要许多因素的组合网络。心肌限制性同源异型盒蛋白Csx/Nkx2.5（Csx）在心前中胚层以及胚胎和成人心脏中表达。Csx的靶向破坏由于心脏形态发生异常而导致胚胎死亡。锌指转录因子GATA 4也在心脏中表达，并已被证明是心管形成所必需的。已知GATA 4激活许多心脏组织限制性基因。在这项研究中，我们测试了Csx和GATA 4是否物理关联并协同激活靶基因的转录。免疫共沉淀实验表明，Csx和GATA 4关联细胞内。有趣的是，体外蛋白质相互作用研究表明，Csx的同源结构域的螺旋III需要与GATA 4相互作用，并且GATA 4的羧基末端锌指是与Csx相关联所必需的。已知这两个区域直接接触同源DNA序列。心房利钠因子（ANF）的启动子-增强子区域含有几个推定的Csx结合位点和共有GATA 4结合位点。瞬时转染试验表明，Csx可以激活ANF报告基因的表达到相同的程度，GATA 4在DNA结合位点依赖性的方式。Csx和GATA 4的共表达协同激活ANF报告基因表达。突变分析表明，这种协同作用需要两种因子完全保留其转录活性，包括辅因子结合活性。这些结果证明了同源异型蛋白和锌指蛋白相互作用的脊椎动物中的第一个例子，以合作调节靶基因的表达。这种组织限制性转录因子之间的协同作用可能是加强组织特异性发育途径的重要机制。
Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.