Misra Chaitali, Chang Sheng-Wei, Basu Madhumita, Huang Nianyuan, Garg Vidu
Center for Cardiovascular and Pulmonary Research and The Heart Center, Nationwide Children's Hospital.
Center for Cardiovascular and Pulmonary Research and The Heart Center, Nationwide Children's Hospital, Department of Pediatrics and Department of Molecular Genetics, The Ohio State University, Columbus, OH 43205, USA
Hum Mol Genet. 2014 Oct 1;23(19):5025-35. doi: 10.1093/hmg/ddu215. Epub 2014 May 8.
Mutations in GATA4 and TBX5 are associated with congenital heart defects in humans. Interaction between GATA4 and TBX5 is important for normal cardiac septation, but the underlying molecular mechanisms are not well understood. Here, we show that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle, but after E15.5, ventricular expression of Tbx5 decreases. Co-localization and co-immunoprecipitation studies demonstrate an interaction of Gata4 and Tbx5 in the developing atria and ventricles, but the ventricular interaction declines after E14.5. Gata4(+/-);Tbx5(+/-) mouse embryos display decreased atrial and ventricular myocardial thickness at E11.5, prior to cardiac septation. To determine the cell lineage in which the interaction was functionally significant in vivo, mice heterozygous for Gata4 in the myocardium or endocardium and heterozygous for Tbx5 (Gata4(MyoDel/wt);Tbx5(+/-) and Gata4(EndoDel/wt);Tbx5(+/-), respectively) were generated. Gata4(MyoDel/wt);Tbx5(+/-) mice displayed embryonic lethality, thin myocardium with reduced cell proliferation, and atrioventricular septation defects similar to Gata4;Tbx5 compound heterozygotes while Gata4(EndoDel/wt);Tbx5(+/-) embryos were normal. Cdk4 and Cdk2, cyclin-dependent kinases required for myocardial development and septation were reduced in Gata4(+/-);Tbx5(+/-) hearts. Cdk4 is a known direct target of Gata4 and the regulation of Cdk2 in the developing heart has not been studied. Chromatin immunoprecipitation and transactivation studies demonstrate that Gata4 and Tbx5 directly regulate Cdk4 while only Tbx5 activates Cdk2 expression. These findings highlight the mechanisms by which disruption of the Gata4 and Tbx5 interaction in the myocardium contributes to cardiac septation defects in humans.
GATA4和TBX5的突变与人类先天性心脏缺陷有关。GATA4和TBX5之间的相互作用对正常心脏分隔很重要，但其潜在的分子机制尚未完全了解。在这里，我们表明Gata4和Tbx5在胚胎心房和心室中共同表达，但在E15.5之后，Tbx5的心室表达下降。共定位和免疫共沉淀研究表明Gata4和Tbx5在发育中的心房和心室中相互作用，但在E14.5之后心室中的相互作用减弱。Gata4(+/-);Tbx5(+/-)小鼠胚胎在E11.5时，即在心脏分隔之前，心房和心室心肌厚度就已降低。为了确定在体内该相互作用发挥功能作用的细胞谱系，构建了心肌或心内膜中Gata4为杂合子且Tbx5为杂合子的小鼠（分别为Gata4(MyoDel/wt);Tbx5(+/-)和Gata4(EndoDel/wt);Tbx5(+/-)）。Gata4(MyoDel/wt);Tbx5(+/-)小鼠表现出胚胎致死性，心肌变薄且细胞增殖减少，以及与Gata4;Tbx5复合杂合子相似的房室间隔缺损，而Gata4(EndoDel/wt);Tbx5(+/-)胚胎则正常。Gata4(+/-);Tbx5(+/-)心脏中参与心肌发育和分隔的细胞周期蛋白依赖性激酶Cdk4和Cdk2减少。Cdk4是已知的Gata4直接靶标，而发育中心脏中Cdk2的调控尚未得到研究。染色质免疫沉淀和反式激活研究表明，Gata4和Tbx5直接调控Cdk4，而只有Tbx5激活Cdk2的表达。这些发现突出了心肌中Gata4和Tbx５相互作用的破坏导致人类心脏分隔缺陷的机制。
