Cicchini Carla, de Nonno Valeria, Battistelli Cecilia, Cozzolino Angela Maria, De Santis Puzzonia Marco, Ciafrè Silvia Anna, Brocker Chad, Gonzalez Frank J, Amicone Laura, Tripodi Marco
Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy.
Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Hematology, Section of Molecular Genetics, Sapienza University of Rome, Rome, Italy.
Biochim Biophys Acta. 2015 Aug;1849(8):919-29. doi: 10.1016/j.bbagrm.2015.05.005. Epub 2015 May 21.
Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B.
Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity.
HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR-29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype.
HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels.
上皮-间质转化（EMT）和相反的间质-上皮转化（MET）是细胞可塑性的表现，这意味着动态且深刻的基因表达重编程。虽然DNA甲基化保证了控制整个转录谱协调调控的主要表观遗传密码，但EMT/MET动态过程中DNA甲基转移酶（DNMT）的活性仍未得到充分研究。在此，我们研究了将MET的主要效应因子HNF4α与DNMT 3A和3B的从头甲基化调控直接联系起来的分子机制。
通过RT-qPCR、蛋白质印迹和免疫细胞化学分析评估EMT/MET标志物、microRNA29和DNMT3s表达之间的相关性。通过抗miR、microRNA前体和化学抑制剂测试microRNA和DNMT3s的功能作用。利用染色质免疫沉淀法研究HNF4α的DNA结合活性。
在体外分化的肝细胞以及体内肝细胞特异性Hnf4α基因敲除小鼠中，HNF4α沉默足以诱导DNMT3B的正向调节，在体外还可诱导DNMT3A，但不能诱导DNMT1。在探索这些观察结果背后的分子机制时，已收集到以下证据：（i）DNMT3水平与其调节因子miR-29a和miR-29b的表达呈负相关；（ii）HNF4α作为miR-29a-b转录的直接调节因子的作用。值得注意的是，在TGFβ诱导的EMT过程中，已证明DNMT3s具有关键作用，因此表明在维持分化表型时需要抑制这些DNMT。
HNF4α通过调节miR-29a和-29b的表达来维持肝细胞特性，而miR-29a和-29b又通过限制DNMT3A和DNMT3B的水平来控制表观遗传修饰。
