Roles of Glycoslyation in Notch Signaling

描述(申请人提供)：当细胞表面的Notch受体被相对细胞上的Notch配体刺激，导致针对众多下游基因的Notch受体胞内域的释放时，Notch信号就会发生。在哺乳动物的发育和分化过程中，许多细胞命运的决定依赖于调控的Notch信号。因此，人类的几种疾病和癌症都是由Notch信号通路的故障引起的。疾病范围从骨骼畸形到心脏病，由Notchl基因突变引起的一种主要癌症是T细胞白血病。O-岩藻糖聚糖结合在Notch受体的EGF重复序列上，在Notch信号的调节中发挥着关键但不明确的作用。在没有它们的情况下，小鼠胚胎在怀孕中期死亡，典型的缺陷是通过所有四个Notch受体失去信号。Notch受体上的O-岩藻糖通过三种边缘GlcNAc转移酶中的一种加入GlcNAc而延长。基因表达异常导致的Notch信号异常与发育缺陷和肿瘤预后有关。因此，了解O-岩藻糖聚糖和不同的边缘活性调控Notch信号的分子机制是非常重要的。然而，这是一个挑战，因为这三个边缘基因经常是共同表达的。我们建议通过产生胚胎、胚胎干细胞(ES)和小鼠胚胎成纤维细胞(MEF)来简化这种情况，这些胚胎、胚胎干细胞和小鼠胚胎成纤维细胞(MEF)从其内源基因或来自Lfng基因的位置表达单一的Fringer基因，或者根本不表达Frage基因。我们将使用这些生物材料，在这些材料中，Notch受体将只携带O-岩藻糖或O-岩藻糖，而GlcNAc由单一的边缘酶转移，以确定每个边缘糖基转移酶在胚胎发育以及T和B细胞发育中的作用。具体目标1将研究只表达Lfng、只表达Mfng或只表达Rfng的小鼠或胎肝细胞的T和B细胞发育和免疫反应。具体目的2是通过重组酶介导盒交换，产生只表达单一FNG活性的小鼠或胚胎。我们将确定由每一个条纹基因编码的GlcNAc-转移酶活性是否能够在体内正确的时间和地点以受调控的方式表达时具有同等的功能。具体目的3是确定哺乳动物Notch受体在胚胎、ES和MEF细胞中单独携带O-岩藻糖信号的机制，以及分别对配体结合和恢复每个条纹活性的信号的影响。联合实验将确定Lfng、Mfng和Rfng的特定作用，并为机制研究创造有价值的小鼠品系和细胞系。

DESCRIPTION (provided by applicant): Notch signaling occurs when cell surface Notch receptors are stimulated by Notch ligands on an apposing cell leading to release of the Notch receptor intracellular domain which targets numerous downstream genes. A large variety of cell fate decisions depend on regulated Notch signaling during development and differentiation in mammals. Thus, several human diseases and cancers arise from malfunctioning of Notch signaling pathways. Diseases range from skeletal deformities to heart disease and a major cancer arising from mutations in Notchl is T cell leukemia. The O-fucose glycans attached to the EGF repeats of Notch receptors play critical but ill-defined roles in the regulation of Notch signaling. In their absence mouse embryos die at mid-gestation with defects typical of a loss of signaling through all four Notch receptors. O-fucose on Notch receptors is elongated by the addition of GlcNAc by one of three Fringe GlcNAc-transferases. Dysregulation of Notch signaling by altered Fringe gene expression has been associated with developmental defects and cancer prognosis. Therefore it is very, important to understand molecular mechanisms by which O-fucose glycans and the different Fringe activities regulate Notch signaling. This is a challenge however, because the three Fringe genes are often co-expressed. We propose to simplify the situation by generating embryos, embryonic stem (ES) cells and mouse embryo fibroblasts (MEF) that express a single Fringe gene from its endogenous locus, or from the Lfng locus, or that express no Fringe genes at all. We will use these biological materials in which Notch receptors will carry only O- fucose or O-fucose with GlcNAc transferred by a single Fringe enzyme, to identify roles for each Fringe glycosyltransferase in embryonic development and in T and B cell development. In Specific aim 1 T and B cell development and immune respones will be investigated in mice or fetal liver cells from embryos that express only Lfng, only Mfng or only Rfng. Specific aim 2 is to generate mice or embryos expressing only a single Fng activity knocked in to the well-characterized Lfng locus by recombinase mediated cassette exchange. We will determine whether the GlcNAc-transferase activity encoded by each Fringe gene is able to function equivalently when expressed in a regulated fashion at the right time and place in vivo. Specific aim 3 is to identify mechanisms by which mammalian Notch receptors carrying solely O-fucose signal in embryos, ES and MEF cells, and the effects on ligand binding and signaling of restoring each Fringe activity separately. The combined experiments will identify specific roles for Lfng, Mfng and Rfng and generate valuable mouse strains and cell lines for mechanistic studies.