Functional Analysis of LEM-domain Nuclear Proteins

Emerin是一种核膜蛋白，可形成板层细丝。Emerin突变导致Emery-Dreifuss肌营养不良症，影响心脏、骨骼肌和肌腱。Emerin属于保守的核蛋白LEM结构域家族。Emerin的LEM结构域与BAF(自整合因子屏障)结合，BAF是一种功能未知的保守染色质蛋白。我们认为LEM蛋白在结构上将染色质(通过BAF)连接到板层，也结合了介导DNA复制或转录抑制的伙伴。我们建议确定线虫中三个保守的LEM蛋白的基本功能：Emerin，MAN1和Lem-3。在埃尔根线虫中，所有或大部分细胞都表达Emerin和MAN1，但RNAi诱导的Emerin或MAN1的缺失没有表型。然而，这两种蛋白质的丢失在胚胎中是致命的。因此，Emerin和MAN1具有必要的功能(S)。我们将检验这三个LEM蛋白都直接与BAF和Lamin结合的假设，并使用定点突变来定位它们的结合区。我们将使用RNAi来耗尽每个LEM蛋白，并使用成对的LEM蛋白来测试体内重叠和细胞类型特定的功能。通过鉴定和表征Emerin、MAN1和lem-3的新结合伙伴，我们将检验我们的假设，即LEM蛋白直接参与复制、转录或层蛋白动力学。我们将确定突变的LEM蛋白，其结合活性在体外定义，是否可以挽救Emerin：MAN1(RNAi)胚胎的致死性。我们将在Emerin缺失或MAN1缺失的背景下筛选合成致命的突变，以确定介导Emerin和MAN1基本功能的蛋白质。这项工作将是第一次对核层进行基因分析。我们的工作将产生关于LEM蛋白功能的基本知识，以及它们与Lamins和BAF的相互作用。这项工作可以扩展到人类，以预测由Emerin丢失引起的心脏病和肌营养不良的分子机制。

Emerin is a nuclear membrane protein that bines lamina filaments. Emerin mutations cause Emery-Dreifuss muscular dystrophy, affecting heart, skeletal muscle, and tendons. Emerin belongs to the conserved LEM-domain family of nuclear proteins. The LEM-domain of emerin binds BAF (barrier to autointegration factor), a conserved chromatin protein of unknown function. We propose that LEM proteins structurally link chromatin (via BAF) to the lamina, and also bind partners that mediate DNA replication or transcriptional repression. We propose to determine the essential function of three conserved LEM proteins in C.elegans: emerin, MAN1 and lem-3. In C. elelgans, all or most cells express emerin and MAN1, but the RNAi-induced loss of emerin or MAN1 has no phenotype. However, loss of both proteins is lethal in embryos. Thus, emerin and MAN1 have essential function(s). We will test the hypothesis that all three LEM proteins bind directly to BAF and lamin, and use site-directed mutagenesis to map their binding regions. We will use RNAi to deplete each LEM protein, and pairs of LEM proteins to test for overlapping and cell-type-specific functions in vivo. By identifying and characterizing new binding partners for emerin, MAN1 and lem- 3, we will test our hypothesis that LEM proteins are directly involved in replication, transcription, or lamin dynamics. We will determine if mutant LEM proteins, whose binding activities are defined in vitro, can rescue lethality of emerin: MAN1 (RNAi) embryos. WE will screen for mutations that are synthetically lethal in an emerin null or MAN1 null background, to identify proteins that mediate the essential functions of emerin and MAN1. This work will be the first genetic analysis of the nuclear lamina. Our work will yield basic knowledge about the functions of LEM proteins, and their interactions with lamins and BAF. This work can be extended to humans to predict the molecular mechanisms of heart disease and muscular dystrophy caused by loss of emerin.