Regulation of nuclear envelope assembly and disassembly

描述(由申请人提供)：核膜蛋白和A型层粘连蛋白细丝的功能，与越来越多的信号通路、人类疾病和人类衰老有关，是生物学中一个重要的开放前沿。我们建议将重点放在Emerin，一个保守的层蛋白结合核膜LEM结构域蛋白，以及它保守的组蛋白结合染色质伙伴屏障-自整合因子(BAF)。人类男性中Emerin的丢失会导致X连锁隐性Emery-Dreifuss肌营养不良症(EDMD)。然而，Emerin在几乎所有组织中都有表达，这表明它在细胞生理学中发挥着更广泛的作用。我们最近从HeLa细胞核中分离纯化和鉴定了六个不同的含有Emerin的复合体，支持这样的假设，即在核膜上出现各种“支架”，具有信号传递、染色质沉默、基因调控和核结构等功能。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损伤的反应减少，通过形成磷酸化的H_2AX焦点来检测。我们之前对BAF的研究表明，BAF在染色质结构、有丝分裂染色体分离、核组装和包括肌肉在内的特定组织中的基因调控中发挥着重要和广泛的作用。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联，包括CUL4-DDB-ROC E3连接酶复合体的聚(ADP-核糖)聚合酶1(PARP1)和CUL4。PARP1和CUL4-DDB-ROC复合体都能修饰核小体组蛋白以响应DNA损伤。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损伤的反应中发挥着广泛的生理作用。目的1通过鉴定BAF对单核小体和重组的12聚体核小体阵列的影响，以及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在下调Emerin或BAF的细胞DNA损伤反应通路中的作用。AIM 3是对线虫BAF功能的全基因组和启动子特异性分析，有望识别与其在肌肉和DNA损伤反应中所起作用相关的基因。公共卫生相关性拟议的工作与了解Emery-Dreifuss肌营养不良症的分子机制有关，该综合征影响三个组织，包括骨骼肌、肌腱和心脏传导系统。对BAF与染色质和组蛋白修饰酶相互作用的拟议研究也与理解为什么BAF对于人类免疫缺陷病毒1型(HIV-1)整合到人类染色体上是必不可少的。拟议中的Emerin和BAF在细胞对DNA损伤的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.