GATA Factor Function in Trophoblast

描述(申请人提供)：滋养层细胞谱系形成胎盘的外部上皮成分，并提供胎儿和母亲之间的功能桥梁。多种转录因子参与了包括滋养层干细胞在内的滋养层细胞亚型的发育和功能。例如，转录因子尾型同源盒2(CDX2)在滋养外胚层的形成和TS细胞的增殖中起着重要的作用。有趣的是，转录因子GATA2和GATA3也参与了滋养层细胞特异性基因表达的转录调控。然而，GATA因子在滋养层细胞系中发挥作用的分子机制尚不清楚。尽管在GATA2-/-和GATA3-/-小鼠中，几个胎盘基因的表达减少，但由于缺乏明显的胎盘表型，导致预测这两个因子在胎盘发育过程中可能以互补的方式发挥作用。然而，实验还没有在GATA2和GATA3都有限的情况下进行。此外，GATA因子在滋养层细胞系分化过程中的表达模式、它们在内源性染色质区域的作用方式以及它们在滋养层细胞中的靶基因还知之甚少。我们发现了GATA3的一个新功能，它选择性地在发育中的小鼠胚胎的滋养外胚层(TE)中表达，调节TE谱系中关键基因的表达，并参与桑椹胚到囊胚的转化。此外，我们还证明，在滋养层巨细胞分化过程中，GATA3和GATA2之间染色质占位的转换是包括CDX2在内的GATA靶基因转录调控的重要机制。因此，我们将利用新的策略来阐明GATA因子在滋养层细胞分化和功能中的作用。提出了三个具体目标。目的1验证GATA因子在滋养层细胞系发育过程中调节CDX2表达的假设。我们将通过检测GATA2和GATA3在植入前小鼠胚胎中的表达模式，通过识别滋养层细胞内源CDX2染色质结构域的GATA因子结合区，以及通过产生基因组CDX2报告来检验这一点。目的研究GATA因子在体外和体内滋养层细胞发育和分化中的作用。我们将验证GATA2和GATA3的缺失将损害滋养层细胞系分化的假设。我们将通过建立一个TS细胞系统来测试这一点，在该系统中，GATA2和GATA3都将是限制性的(体外方法)，并在体内通过RNA干扰在滋养层细胞系中击倒GATA2和GATA3。在目标3中，我们将使用染色质免疫沉淀和基因组微阵列相结合的方法，检测GATA2和GATA3在滋养层细胞中是否有共同和独特的靶基因。我们还将确定GATA3是否具有分化阶段特异的靶基因。

DESCRIPTION (provided by applicant): Trophoblast cell lineages form the outer epithelial component of the placenta and provide the functional bridge between the fetus and the mother. Multiple transcription factors have been implicated in the development and function of trophoblast cell subtypes including trophoblast stem (TS) cells. For example, transcription factor caudal-type homeobox 2 (CDX2) is important for the derivation of trophectoderm and proliferation of TS cells. Interestingly, transcription factors, GATA2 and GATA3, have also been implicated in the transcriptional regulation of trophoblast cell-specific gene expression. However, molecular mechanisms of GATA factor function in trophoblast cell lineages are poorly understood. Although the expression of several placental genes is reduced in Gata2-/- and Gata3-/- mice, the lack of an overt placental phenotype led to the prediction that these two factors might function in a complementary fashion during placental development. However, experiments have not been done in a context where both GATA2 and GATA3 are limiting. In addition, GATA factor expression pattern during trophoblast lineage differentiation, their mode of function in endogenous chromatin domains, and their target genes in trophoblast cells are poorly understood. We identified a novel function of GATA3, in which it is selectively expressed in the trophectoderm (TE) of developing mouse embryo, regulates expression of key genes in TE lineage, and is involved in morula to blastocyst transformation. In addition, we demonstrated that, during trophoblast giant cell differentiation, switch in chromatin occupancy between GATA3 and GATA2 is an important mechanism for the transcriptional regulation of GATA target genes including Cdx2. Therefore, we will utilize novel strategies to elucidate role of GATA factors in trophoblast cell differentiation and function. Three specific aims are proposed. Aim 1 will test the hypothesis that GATA factors regulate Cdx2 expression during development of the trophoblast lineage. We will examine this by determining GATA2 and GATA3 expression pattern in pre-implantation mice embryos, by identifying GATA factor binding regions at the endogenous Cdx2 chromatin domain in trophoblast cells, and by generating a genomic Cdx2 reporter. Aim 2 will determine the role of GATA factors during trophoblast cell development and differentiation both in vitro and in vivo. We will test the hypothesis that loss of both GATA2 and GATA3 will impair trophoblast lineage differentiation. We will test this by establishing a TS cell system, in which both GATA2 and GATA3 will be limiting (in vitro approach), and by knocking-down GATA2 and GATA3 by RNA interference in the trophoblast cell lineage in vivo. In aim 3, using a chromatin immunoprecipitation coupled with genomic microarray approach, we will test whether GATA2 and GATA3 have common and unique target genes in trophoblast cells. We will also determine whether GATA3 have differentiation stage specific target genes.