Roles of Glycoslyation in Notch Signaling

描述（由申请人提供）：当细胞表面Notch受体被并置细胞上的Notch配体刺激时，Notch信号传导发生，导致靶向许多下游基因的Notch受体胞内结构域释放。在哺乳动物的发育和分化过程中，各种各样的细胞命运决定取决于受调节的Notch信号传导。因此，几种人类疾病和癌症是由Notch信号通路的功能障碍引起的。疾病的范围从骨骼畸形到心脏病，并且由Notchl突变引起的主要癌症是T细胞白血病。附着在Notch受体的EGF重复序列上的O-岩藻糖聚糖在Notch信号传导的调节中起着关键但不明确的作用。在缺乏它们的情况下，小鼠胚胎在妊娠中期死亡，具有典型的缺陷，即通过所有四种Notch受体的信号丢失。Notch受体上的O-岩藻糖通过三种Fringe GlcNAc转移酶之一添加GlcNAc而延长。由改变的Fringe基因表达引起的Notch信号转导失调与发育缺陷和癌症预后相关。因此，理解O-岩藻糖聚糖和不同的Fringe活性调节Notch信号传导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共同表达。我们建议通过产生胚胎、胚胎干（ES）细胞和小鼠胚胎成纤维细胞（MEF）来简化这种情况，所述胚胎、胚胎干细胞和小鼠胚胎成纤维细胞从其内源基因座或从Lfng基因座表达单个Fringe基因，或者根本不表达Fringe基因。我们将使用这些生物材料，其中Notch受体将仅携带O-岩藻糖或O-岩藻糖与由单个Fringe酶转移的GlcNAc，以鉴定每个Fringe糖基转移酶在胚胎发育以及T和B细胞发育中的作用。在具体目标1中，将在仅表达Lfng、仅表达Mfng或仅表达Rfng的小鼠或来自胚胎的胎肝细胞中研究T和B细胞发育和免疫应答。具体目标2是产生仅表达通过重组酶介导的盒交换敲入充分表征的Lfng基因座的单一Fng活性的小鼠或胚胎。我们将确定是否GlcNAc-转移酶活性编码的每一个边缘基因是能够发挥同等作用时，在一个受管制的方式在正确的时间和地点在体内表达。具体目标3是确定哺乳动物Notch受体在胚胎、ES和MEF细胞中仅携带O-岩藻糖信号的机制，以及分别恢复每种Fringe活性对配体结合和信号传导的影响。组合实验将鉴定Lfng、Mfng和Rfng的特定作用，并产生有价值的小鼠品系和细胞系用于机制研究。

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.