Combinatorial expression of GATA4, Nkx2-5, and serum response factor directs early cardiac gene activity
Combinatorial expression of GATA4, Nkx2-5, and serum response factor directs early cardiac gene activity
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DOI：
10.1074/jbc.m203122200
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发表时间：
2002-07-12
影响因子：
4.8
通讯作者：
Schwartz, RJ
中科院分区：
文献类型：
--
作者：
Sepulveda, JL;Vlahopoulos, S;Schwartz, RJ
在此，血清反应因子（SRF）的限制性表达与早期鸡胚中的Nkx 2 -5和GATA 4转录物密切重叠，与心脏α-肌动蛋白（α CA）转录物和新生心肌细胞的最早出现相一致。在异源CV1成纤维细胞和Schneider 2昆虫细胞中SRF（MADS盒因子Nkx 2 -5（NK 4同源结构域））和/或GATA 4（双C4锌指蛋白）的组合表达证明了alphaCA启动子活性的协同诱导。这三种因子在鼠胚胎干细胞中诱导内源性alphaCA mRNA超过100倍。此外，DNA结合缺陷型突变体N-kx 2 - 5 pm在SRF和GATA 4存在的情况下，在所有四种SRE存在的情况下，有效地共激活了alphaCA启动子，并且当单个SRE突变和/或连续缺失时，其显著减弱。相反，SRFpm，SRF DNA结合突变体的引入，阻断了所有的alphaCA启动子结构的激活。这些测定表明，依赖于协同SRF结合，以促进Nkx 2 -5和GATA 4募集到alphaCA启动子。此外，观察到通过SRF募集Nkx 2 -5和GATA 4强烈增强SRF DNA结合亲和力。这种机制允许形成更高级的alphaCA启动子DNA结合复合物，导致SRF与Nkx 2 -5和GATA 4物理缔合的模型。
Herein, the restricted expression of serum response factors (SRF) closely overlapped with Nkx2-5 and GATA4 transcripts in early chick embryos coinciding with the earliest appearance of cardiac alpha-actin (alphaCA) transcripts and nascent myocardial cells. The combinatorial expression of SRF, a MADS box factor Nkx2-5 (a NK4 homeodomain), and/or GATA4, a dual C4 zinc finger protein, in heterologous CV1 fibroblasts and Schneider 2 insect cells demonstrated synergistic induction of alphaCA promoter activity. These three factors induced endogenous alphaCA mRNA over a 100-fold in murine embryonic stem cells. In addition, the DNA-binding defective mutant N-kx2-5pm efficiently coactivated the alphaCA promoter in the presence of SRF and GATA4 in the presence of all four SREs and was substantially weakened when individual SREs were mutated and or serially deleted. In contrast, the introduction of SRFpm, a SRF DNA-binding mutant, blocked the activation with all of the alphaCA promoter constructions. These assays indicated a dependence upon cooperative SRF binding for facilitating the recruitment of Nkx2-5 and GATA4 to the alphaCA promoter. Furthermore, the recruitment of Nkx2-5 and GATA4 by SRF was observed to strongly enhance SRF DNA binding affinity. This mechanism allowed for the formation of higher ordered alphaCA promoter DNA binding complexes, led to a model of SRF physical association with Nkx2-5 and GATA4.