Screening for genes essential for develop of the mouse

我们正在使用两种方法来识别和分离与哺乳动物胚胎和胎儿的调节生长和分化有关的基因。第一种是插入突变，即通过将外源DNA插入到基因中而产生突变表型。这也在突变的位置创建了一个分子标签，提供了对基因的直接访问。从组织培养中故意感染逆转录病毒的胚胎干细胞衍生的转基因小鼠品系中筛选出4个隐性产前致死插入突变。其中之一是节点基因的突变。另一个是相扑/先锋蛋白特异蛋白酶1(SENP1)基因的突变，这是最近发现的一种参与相扑调节的蛋白质。相扑是一种与泛素相关的小蛋白，通过类似泛素化的过程添加到蛋白质中。SENP1起到从修饰蛋白质中去除SUMO的作用。SENP1基因的插入突变导致纯合子中约90%的表达水平下降。与这种脱合酶水平降低相一致的是，纯合子突变胚胎中的总合蛋白水平显著增加。纯合子胚胎在中期由于胎盘缺陷而死亡，这表明相扑修改在胚胎外发育中起着至关重要的作用。 作为识别发育重要基因的另一种方法，我们假设许多发育调节蛋白的活性可能受到泛素介导的蛋白质降解的调节，并且这些分子可以通过它们与E3泛素连接酶的相互作用来识别，E3泛素连接酶是泛素化途径的组成部分，具有特异性。作为鉴定这些蛋白质的第一步，我们使用发育调节的E3Nedd-4在酵母双杂交筛选中表达在中期妊娠小鼠胚胎中的基因。这使得我们能够分离Nedd-4结合蛋白(N4BP)1-4。N4BP-1、-2和-3是由新基因编码的，而NBP4是一个已知的蛋白质PLIC2。NEDD-4在体外可使N4BP-1和N4BP-2泛素化。我们现在已经证明了N4BP-2在体内是多泛素化的，并且在不同的蛋白酶体抑制剂处理的细胞中，N4BP-2的稳态水平增加。这一结果表明N4BP-2是Nedd-4介导的泛素化和蛋白酶体降解的真实底物，并为该方法能够在蛋白质稳定水平上识别调控发育的蛋白质提供了原理证据。相反，我们发现N4BP-1在体内是单一泛素化的，并且Nedd-4水平的增加增强了单一泛素化的水平。单一泛素化不是蛋白质降解的信号，而是调节蛋白质的位置和活性。N4BP-1转基因细胞的免疫组织化学分析显示，细胞核位于离散的圆形结构中，也含有相扑修饰的蛋白质。大多数核Sumoylated蛋白存在于PML(早幼粒细胞白血病)核小体中，包括PML蛋白本身。PML是一种肿瘤抑制因子，以其Sumoyl化形式可以将其他成分招募到PML体内。PML小体的功能尚不清楚，但它们与细胞凋亡、转录和抗原提呈有关。对N4BP-1转基因细胞的进一步分析表明，PML与N4BP-1在细胞核内共定位。此外，N4BP-1转基因细胞中PML小体的数量和大小均显著增加。我们在N4BP-1上发现了3个一致的苏莫化位点。其中一个基因的突变会导致PML小体的大小急剧增加。综上所述，这些结果表明，N4BP-1是PML小体的一种新成分，与PML本身一样，可以调节它们的组装。

We are using two approaches to identify and isolate genes involved in the regulated growth and differentiation of the mammalian embryo and fetus. The first of these is insertional mutagenesis, in which mutant phenotypes are generated by the insertion of exogenous DNA into a gene. This also creates a molecular tag at the mutant locus, providing direct access to the gene. A screen of transgenic mouse strains derived from embryonic stem (ES) cells deliberately infected in tissue culture with retroviruses identified 4 recessive prenatal lethal insertional mutations. One of these is a mutation of the nodal gene. Another is a mutation of the gene for Sumo/sentrin specific protease 1 (SENP1), a recently identified protein involved in the regulation of Sumoylation. Sumo is a small protein related to ubiquitin that is added to proteins in a process similar to ubiquitination. SENP1 acts to remove Sumo from modified proteins. Insertional mutation of the SENP1 gene leads to an approximately 90% reduction of expression levels in homozygotes. Consistent with reduced levels of this de-Sumoylating enzyme, there is a dramatic increase in the levels of sumoylated proteins in homozygous mutant embryos. Homozygous embryos die around midgestation due to placental defects, indicating an essential role for SUMO modification in extraembryonic development. As another approach to identify developmentally important genes, we hypothesized that the activity of many developmental regulatory proteins might be regulated by ubiquitin-mediated protein degradation, and that these molecules can be identified by their interaction with E3 ubiquitin ligases, the component of the ubiquitylation pathway conferring specificity. As a first step to identify such proteins, we used the developmentally regulated E3 Nedd-4 in a yeast two-hybrid screen of genes expressed in the midgestation mouse embryo. This allowed us to isolate Nedd-4 Binding Proteins (N4BP) 1-4. N4BP-1, -2 and -3 are encoded by novel genes while NBP4 is a known protein, PLIC2. N4BP-1 and -2 can be ubiquitylated in vitro by Nedd-4. We have now demonstrated that N4BP-2 is poly-ubiquitylated in vivo and that steady state levels of N4BP-2 increase in cells treated with different proteasome inhibitors. This result indicates that N4BP-2 is a bona fide substrate for Nedd-4 mediated ubiquitylation and proteasome degradation, and provides proof of principal that this approach can identify developmental proteins regulated at the level of protein stability. In contrast, we found that N4BP-1 is mono-ubiquitylated in vivo and that increased levels of Nedd-4 enhance the level of mono-ubiquitylation. Mono-ubiquitylation is not a signal for protein degradation, rather it may serve to regulate the location and activity of proteins. Immunohistochemical analysis of N4BP-1 transfected cells revealed a nuclear location, in discrete circular structures also containing Sumo-modified proteins. Most nuclear Sumoylated proteins are found in PML (promyelocytic leukemia) nuclear bodies, including the PML protein itself. PML is a tumor suppressor and in its Sumoylated form can recruit other components to PML bodies. The function of PML bodies is not well understood but they have been implicated in apoptosis, transcription and antigen presentation. Further analysis of N4BP-1 transfected cells showed that PML colocalized with N4BP-1 in the nucleus. In addition, the number and size of PML bodies were significantly increased in N4BP-1 transfected cells. We have found 3 consensus Sumoylation sites in N4BP-1. Mutation of one of these leads to a dramatic increase in the size of PML bodies. Together these results suggest that N4BP-1 is a new component of PML bodies and, like PML itself, can regulate their assembly.