转录因子GATA4和Foxa2在调控内胚层细胞肝向分化中的相互作用

Transcription factors play the important roles in guiding the hepatic fate of embryonic endoderm cells by controlling the relative gene transcription events. Study on the activities of some key transcription factors during hepatic differentiation will be helpful for elucidation of mechanism of liver organogenesis. Previously, it has been known that GATA4 and Foxa2, as key transcription factors, could determine the hepatic fate of embryonic endoderm cells during liver organogenesis or hepatogenesis. However, the target genes of two transcription factors during the critical process of hepatogenesis could not be known because there were no sufficient and synchronized embryonic endoderm cells that could be used in molecular study. For the same reason, the proposed hypothesis of GATA4 and Foxa2 as "pioneer factors" during hepatogenesis has not been proved genome-widely. Recently, we have found that the in vitro hepatic differentiation of ES cells could mimic the process of embryonic hepatogenesis. During the hepatic induction process of ES cells, we can generate sufficient ES cell-derived definitive endoderm cells and ES cell-derived hepatic progenitor cells. With enough cells in ChIP-seq analysis, we can use the high though-put DNA sequencing to collectively discover the target genes of both GATA4 and Foxa2. This study will be performed in both ES cell-derived definitive endoderm cells and hepatic progenitor cells, which represent the two critical differentiation time points during hepatogenesis. From the expected results, we will be able to know all target genes for both GATA4 and Foxa2. We will expect to find the novel genes for hepatogenesis, and to genome-widely prove the hypothesis of "pioneer factor" for both GATA4 and Foxa2. In addition, the transcription factor Tcf12 in E-box family was first found in our preliminary ChIP experiments with using both antibodies of both GATA4 and Foxa2. We will determine whether Tcf12 as another regulator involving into the co-operative activities of GATA4 and Foxa2 during hepatogenesis. Our study will be useful for elucidating the molecular mechanism of multiple transcription factors in co-regulation of hepatogenesis, as well as for the induction of stem cell-derived hepatic cells at industrial level.

转录因子GATA4和Foxa2在肝脏器官发生中决定了内胚层细胞向肝细胞分化的命运。然而，由于不能从胚胎组织中分离到足够数量、发育同步的内胚层细胞和肝前体细胞，长期以来无法实施对GATA4和Foxa2下游靶基因的研究，也无法在全基因组水平证明GATA4和Foxa2的先锋因子作用。胚胎干细胞的肝向诱导分化可以重演胚胎肝脏器官发生过程，可获得足够的内胚层细胞和肝前体细胞。本课题应用ChIP-Seq技术，在全基因组水平研究Foxa2和GATA4靶基因，以期获得肝向分化中的未知基因，并在全基因组水平上证明Foxa2和GATA4作为先锋因子的表观调控作用。另外，本申请还将研究Tcf12与GATA4和Foxa2互相结合以及结合位点间的相互关系。对GATA4和Foxa2相互作用的认识，有助于阐明其作为关键转录因子在肝向分化中转录调控方面的分子机理，有助于指导干细胞肝向分化向正确和高效的细胞产业化方向发展。

以经过优化的由胚胎干细胞（Embryonic Stem Cell, ESC）经DE阶段分化至早期肝细胞的高效模型为研究基础，我们应用ChIP-Seq技术，在分化得到的DE细胞中，揭示了Foxa2和GATA4在全基因组范围内的结合位点及下游基因。我们发现GATA4和Foxa2的靶基因中有很大一部分是和发育过程相关的基因。进一步的研究分析表明，GATA4和Foxa2的ChIP-Seq数据有很大的重合，这说明在ESC衍生的DE细胞中两者共有很多的靶基因，重合的这部分基因有很大一部分是和肝器官发育过程相关的，其中包括与DE发育相关的基因，如Sox17, Eomes, GATA4, Foxa2, Gsc, Hhex,以及与早期肝脏发育相关的基因,如Afp, Ttr, Timd2。随后的Sequential ChIP实验也表明了，在所诱导获得的DE细胞中，GATA4和Foxa2确实共同结合上述几个共有靶基因的调控区。 另外，GATA4和Foxa2的共结合在Afp和Ttr上还表现出了进一步的共调控关系，两者的正常表达对于DE细胞向肝细胞分化时Afp和Ttr的表达是必需的。由此推测，在DE细胞中GATA4和Foxa2共调控一些DE发育及肝脏发生相关的靶基因。最后，根据ChIP-Seq数据分析和ChIP等实验发现，Tcf12，一个E-box家族的转录因子，作为一个新的结合伙伴，在DE细胞中广泛参与GATA4和Foxa2对靶基因的结合，并由此推测GATA4，Foxa2和Tcf12可能相互协作，共同调控DE的肝向分化。此项研究中，首次实现关于肝向分化过程中转录因子Foxa2和GATA4在全基因组范围内转录调控的探索，并且为胚胎发育中的肝脏发生的机理研究和ESC肝向分化两方面的再生医学应用研究都提供了非常宝贵的信息。