Roles of Glycoslyation in Notch Signaling

描述（由申请人提供）：当细胞表面的Notch受体受到相邻细胞上的Notch配体的刺激，导致Notch受体在细胞内区域释放，靶向许多下游基因时，Notch信号就会发生。在哺乳动物的发育和分化过程中，大量的细胞命运决定依赖于Notch信号的调节。因此，一些人类疾病和癌症是由Notch信号通路的功能失调引起的。疾病范围从骨骼畸形到心脏病，Notchl突变引起的一种主要癌症是T细胞白血病。附着在Notch受体的EGF重复序列上的O聚焦聚糖在Notch信号的调控中起着关键但不明确的作用。在缺乏这些基因的情况下，小鼠胚胎在妊娠中期死亡，并伴有典型的四种Notch受体信号缺失的缺陷。通过三种边缘GlcNAc转移酶中的一种加入GlcNAc， Notch受体上的O-聚焦被拉长。边缘基因表达改变引起的Notch信号失调与发育缺陷和癌症预后有关。因此，了解O聚焦聚糖和不同边缘活性调控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通常是共同表达的。为了简化这种情况，我们建议从胚胎、胚胎干细胞（ES）和小鼠胚胎成纤维细胞（MEF）的内源性基因座，或从lfg基因座，或根本不表达边缘基因，产生表达单一边缘基因的胚胎、胚胎干细胞（ES）和小鼠胚胎成纤维细胞（MEF）。我们将利用这些Notch受体仅携带O型或O型GlcNAc的生物材料，确定每种Fringe糖基转移酶在胚胎发育和T细胞和B细胞发育中的作用。在特异性目的1中，将研究仅表达lng、mng或rng的小鼠或胚胎肝细胞中T和B细胞的发育和免疫应答。具体目标2是通过重组酶介导的盒式交换产生仅表达一种敲入已知Lfng位点的单一Fng活性的小鼠或胚胎。我们将确定每个Fringe基因编码的glcnac转移酶活性是否能够在适当的时间和地点在体内以受调节的方式表达时发挥同等的功能。具体目的3是确定哺乳动物Notch受体在胚胎、胚胎干细胞和MEF细胞中单独携带O-聚焦信号的机制，以及分别恢复每种边缘活性对配体结合和信号传导的影响。联合实验将确定lfg， mfg和Rfng的特定作用，并为机制研究产生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.