Regulation of nuclear envelope assembly and disassembly

描述（由申请人提供）：核膜蛋白和a型纤层蛋白丝的功能与越来越多的信号通路、人类疾病（“纤层病”）和人类衰老有关，是生物学中一个重要的开放前沿。我们建议将重点放在emerin上，这是一种保守的核膜结合lem结构域蛋白，以及它的保守的基本组蛋白结合染色质伴侣-自整合障碍因子（BAF）。人类男性emerin缺失导致x连锁隐性emeri - dreifuss肌营养不良症（EDMD）。然而，emerin几乎在所有组织中表达，表明在细胞生理学中有更广泛的作用。我们最近从HeLa细胞核中纯化和表征了六种不同的含emerin复合物，支持了emerin在核膜上“支架”多种多蛋白复合物的假设，其功能包括信号传导、染色质沉默、基因调控和核结构。有趣的是，emerin在体内与DNA损伤反应激酶（DNA- pk）相互作用，并且emerin下调的HeLa细胞对DNA损伤的反应降低，通过磷酸化- h2ax灶的形成进行了检测。我们之前对BAF （emerin和所有其他lem结构域核蛋白的共同伴侣）的研究表明，在包括肌肉在内的特定组织中，BAF在染色质结构、有丝分裂染色体分离、核组装和基因调控中具有重要而广泛的作用。我们的支持结果表明，BAF与核小体相互作用，在体内影响几种不同的组蛋白翻译后修饰，并在体内与至少两种组蛋白修饰蛋白相关，包括聚（adp -核糖）聚合酶1 （PARP1）和Cul4- ddb - roc E3连接酶复合物的Cul4。PARP1和Cul4-DDB- ROC复合物在DNA损伤时都会修饰核小体组蛋白。这些发现表明BAF是肌肉基因表达的调节因子（因此与EDMD疾病机制相关），并表明BAF和emerin在细胞对DNA损伤的反应中具有广泛的生理作用。目的1将通过表征BAF对单核小体和重组的12聚核小体阵列的影响，并通过确定BAF对parp1介导的多核小体折叠的影响，来验证BAF致密核小体的假设。目的2将描述emerin和BAF在emerin或BAF下调的细胞DNA损伤反应通路中的作用。目的3是对秀丽隐杆线虫中BAF功能的全基因组和启动子特异性分析，预计将鉴定其在肌肉和DNA损伤反应中作用的相关基因。拟议的工作与了解Emery- Dreifuss肌营养不良症的分子机制有关，Emery- Dreifuss肌营养不良症是一种影响骨骼肌、肌腱和心脏传导系统等三种组织的综合征。对BAF与染色质和组蛋白修饰酶相互作用的研究也有助于理解为什么BAF是人类免疫缺陷病毒1型（HIV- 1）整合到人类染色体中所必需的。新兴蛋白和BAF在细胞对DNA损伤反应中的研究与癌症和人类衰老有关。

DESCRIPTION (provided by applicant): The functions of nuclear membrane proteins and A-type lamin filaments, linked to a growing number of signaling pathways, human diseases (`laminopathies') and human aging, are an important open frontier in biology. We propose to focus on emerin, a conserved lamin-binding nuclear membrane LEM-domain protein, and its conserved essential histone-binding chromatin partner Barrier-to-Autointegration Factor (BAF). Loss of emerin in human males causes X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD). However, emerin is expressed in nearly all tissues, suggesting wider roles in cell physiology. Our recent purification and characterization of six distinct emerin-containing complexes from HeLa cell nuclei support the hypothesis that emerin `scaffolds' a variety of multi-protein complexes at the nuclear envelope, with functions that include signaling, chromatin silencing, gene regulation and nuclear architecture. Interestingly, emerin interacts with the DNA damage-response kinase, DNA-PK, in vivo, and emerin-downregulated HeLa cells have reduced response to DNA damage, as assayed by the formation of phosphorylated-H2AX foci. Our previous studies of BAF, the shared partner for emerin and all other LEM-domain nuclear proteins, demonstrated essential and broad-ranging roles in chromatin structure, mitotic chromosome segregation, nuclear assembly and gene regulation in specific tissues including muscle. Our supporting results show that BAF interacts with nucleosomes, influences several different histone posttranslational modifications in vivo, and associates in vivo with at least two histone-modifying proteins including poly(ADP-ribose) polymerase 1 (PARP1) and Cul4 of the Cul4-DDB-ROC E3 ligase complex. Both PARP1 and the Cul4-DDB- ROC complex modify nucleosomal histones in response to DNA damage. These findings implicate BAF as a regulator of muscle gene expression (and therefore relevant to the EDMD disease mechanism), and suggest broad physiological roles for BAF and emerin during cellular responses to DNA damage. Aim 1 will test the hypothesis that BAF compacts nucleosomes by characterizing the effects of BAF on mono-nucleosomes and reconstituted 12-mer nucleosome arrays, and by determining the effect of BAF on PARP1-mediated polynucleosome folding in vitro. Aim 2 will characterize roles for emerin and BAF in DNA damage response pathways in cells downregulated for emerin or BAF. Aim 3 is a whole-genome and promoter-specific analysis of BAF function in C. elegans, which is expected to identify genes relevant to its roles in muscle and DNA- damage responses. PUBLIC HEALTH RELEVANCE The proposed work is relevant to understanding the molecular mechanisms of Emery- Dreifuss muscular dystrophy, a syndrome that affects three tissues including skeletal muscle, tendons and the cardiac conduction system. The proposed investigation of BAF's interactions with chromatin and histone-modifying enzymes are also relevant to understanding why BAF is essential for human immunodeficiency virus type 1 (HIV- 1) to integrate into human chromosomes. The proposed studies of emerin and BAF in cellular responses to DNA damage are relevant to cancer and human aging.