Screening for genes essential for development of the mou

我们正在使用两种方法来确定和分离基因参与调节生长和分化的哺乳动物胚胎和胎儿。第一种是插入诱变，其中通过将外源DNA插入到基因中产生突变表型。这也在突变位点产生了一个分子标签，提供了直接进入基因的途径。用逆转录病毒在组织培养中故意感染胚胎干细胞（ES），筛选转基因小鼠品系，鉴定出4种隐性产前致死插入突变。其中之一是nodal基因的突变。另一个是Sumo/sentrin特异性蛋白酶1（SENP 1）基因的突变，SENP 1是最近鉴定的参与Sumoylation调节的蛋白质。Sumo是一种与泛素相关的小蛋白，它以类似于泛素化的过程被添加到蛋白质中。SENP 1的作用是从修饰的蛋白质中去除Sumo。SENP 1基因的插入突变导致纯合子中表达水平降低约90%。与这种去小泛素化酶的水平降低一致，纯合突变胚胎中小泛素化蛋白的水平显著增加。纯合子胚胎在妊娠中期由于胎盘缺陷而死亡，表明SUMO修饰在胚外发育中起重要作用。 作为鉴定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鉴定，E3泛素连接酶是赋予特异性的泛素化途径的组分。作为第一步，以确定这样的蛋白质，我们使用了发育调节E3 Nedd-4在酵母双杂交筛选的基因表达在妊娠中期的小鼠胚胎。这使我们能够分离Nedd-4结合蛋白（N4 BP）1-4。N4 BP-1、N4 BP-2和N4 BP-3由新基因编码，而NBP 4是已知的蛋白质PLIC 2。N4 BP-1和N4 BP-2可以在体外被Nedd-4泛素化。我们现在已经证明N4 BP-2在体内是聚泛素化的，并且在用不同蛋白酶体抑制剂处理的细胞中N4 BP-2的稳态水平增加。这一结果表明，N4 BP-2是Nedd-4介导的泛素化和蛋白酶体降解的真正底物，并提供了该方法可以鉴定在蛋白质稳定性水平上调节的发育蛋白质的原理证据。相反，我们发现N4 BP-1在体内是单泛素化的，并且Nedd-4水平的增加增强了单泛素化的水平。单泛素化不是蛋白质降解的信号，而是可能用于调节蛋白质的位置和活性。N4 BP-1转染细胞的免疫组织化学分析显示了核的位置，在离散的环状结构也含有Sumo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，其Sumoylated形式可以募集其他成分到PML体。PML小体的功能还不清楚，但它们与细胞凋亡、转录和抗原呈递有关。N4 BP-1转染细胞的进一步分析显示PML与N4 BP-1共定位于细胞核中。此外，在N4 BP-1转染的细胞中，PML小体的数量和大小显著增加。我们在N4 BP-1中发现了3个共有的Sumoylation位点。其中之一的突变导致PML小体的大小急剧增加。总之，这些结果表明，N4 BP-1是PML体的一个新的组成部分，就像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.