GATA Factor Function in Trophoblast

描述（由申请人提供）：滋养细胞系形成胎盘的外上皮成分，并提供胎儿和母亲之间的功能桥梁。多种转录因子参与滋养细胞亚型（包括滋养细胞干细胞）的发育和功能。例如，转录因子尾型同源盒2 （CDX2）对滋养外胚层的衍生和TS细胞的增殖很重要。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因子在滋养细胞谱系中的分子机制尚不清楚。虽然几个胎盘基因的表达在Gata2-/-和Gata3-/-小鼠中减少，但缺乏明显的胎盘表型导致预测这两个因素可能在胎盘发育过程中以互补的方式起作用。然而，在GATA2和GATA3都有限制的情况下，还没有进行实验。此外，GATA因子在滋养细胞谱系分化过程中的表达模式、其在内源性染色质结构域中的功能模式及其在滋养细胞中的靶基因尚不清楚。我们发现了GATA3的一种新功能，它在发育中的小鼠胚胎的滋养外胚层（TE）中选择性表达，调节TE谱系中关键基因的表达，并参与桑葚胚到囊胚的转化。此外，我们证明了在滋养细胞巨细胞分化过程中，染色质占用在GATA3和GATA2之间的转换是GATA靶基因包括Cdx2转录调控的重要机制。因此，我们将利用新的策略来阐明GATA因子在滋养细胞分化和功能中的作用。提出了三个具体目标。目的1将验证GATA因子在滋养细胞谱系发育过程中调节Cdx2表达的假设。我们将通过确定植入前小鼠胚胎中GATA2和GATA3的表达模式，通过鉴定滋养细胞内源性Cdx2染色质区域的GATA因子结合区域，以及通过生成基因组Cdx2报告基因来验证这一点。目的2将确定GATA因子在体外和体内滋养细胞发育和分化过程中的作用。我们将验证GATA2和GATA3的缺失会损害滋养细胞谱系分化的假设。我们将通过建立一个TS细胞系统来测试这一点，其中GATA2和GATA3将被限制（体外方法），并通过RNA干扰在滋养细胞谱系中敲低GATA2和GATA3。在目标3中，我们将使用染色质免疫沉淀结合基因组微阵列方法，测试滋养细胞中GATA2和GATA3是否具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性靶基因。

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. PUBLIC HEALTH RELEVANCE: Early pregnancy loss is a major public health concern. One of the major causes of early pregnancy loss is defective function of the trophectoderm. In addition, defects in placental development due to impaired trophoblast cell functions lead to pregnancies that are at risk for miscarriage and intrauterine growth retardation, and are associated with preeclampsia, a leading cause of maternal death and premature birth. Therefore, understanding the molecular mechanisms of development and function of trophoblast cell lineages is critical to gaining insights into important features of pregnancy loss and pregnancy associated disorders.