Regulation of Nuclear Envelope Assembly and Disassembly

描述(由申请人提供)：我们的目标是了解屏障-自整合因子(BAF)及其保守的结合伙伴Emerin在核膜上的功能。BAF是后生动物中一种高度保守的DNA结合蛋白；没有BAF，分裂的细胞就会死亡。BAF结合一系列核膜蛋白，包括LAP2β和Emerin。Emerin的突变会导致一种组织特异性疾病，Emery-Dreifuss肌营养不良症(EDMD)，它会影响心脏、骨骼肌和肌腱。我们发现Emerin可以直接与包括GCL在内的几个转录抑制因子结合。有趣的是，BAF在体外与GCL竞争与Emerin的结合，提示BAF在体内可能拮抗抑制物与Emerin的结合。我们进一步发现，BAF直接与组蛋白H1和组蛋白H3结合，提示BAF与核小体相互作用，在体内可能调节或受染色质结构的影响。我们认为，Emerin和BAF在核内膜上形成多种蛋白质复合体，影响染色质结构和基因调控。我们发现了一种名为BFL的哺乳动物蛋白质，它与BAF有54%的同源性。BFL与BAF和LAP2β相互作用，但不与DNA或Emerin结合。BFL在大多数组织中都有表达，但在心脏和骨骼肌(选择性地受到EDMD疾病的影响)中没有表达，这表明BFL可能保护其他组织免受Emerin的丢失。我们的BAF、BFL和Emerin的机制和功能模型将通过生化、显微镜、无细胞非洲爪哇提取物和人体细胞系的体内分析进行测试。我们有四个具体目标。(1)确定组蛋白H1和H3中的哪些结构域与BAF结合；检验BAF与核小体相互作用或影响HI介导的染色质压缩的假设；确定BAF与组蛋白H3的结合是否受H3尾部的翻译后修饰调节。(2)确定磷酸化如何影响BAF与Emerin和其他伙伴的结合，并剖析BAF在体外核组装过程中的结构作用。(3)在培养的哺乳动物细胞中使用荧光素酶报告分析来测试Emerin抑制基因表达的假设；我们还将使用我们收集的大量Emerin和BAF的生化特征突变来了解不同的Emerin复合体在体外和活细胞中的功能。(4)在荧光素酶报告实验中使用已证实的转录抑制因子LAP2beta来验证我们的假设，即BFL或BAF在体内调节LAP2β抑制；使用生化竞争分析来确定哪些LAP2β或Emerin结合蛋白相互竞争或合作与LAP2β或Emerin结合。我们的目标是确定哪些伙伴对内核膜上的抑制性LEM结构域蛋白质复合体做出贡献，以及哪些伙伴锚定这些复合体。

DESCRIPTION (provided by applicant): Our goal is to understand the functions of Barrier-to-Autointegration Factor (BAF) and its conserved binding partner emerin at the nuclear envelope. BAF is a highly conserved DNA-binding protein in metazoans; without BAF, dividing cells die. BAF binds a family of nuclear membrane proteins that includes LAP2beta and emerin. Mutations in emerin cause a tissue-specific disease, Emery-Dreifuss muscular dystrophy (EDMD), which affects heart, skeletal muscle and tendons. We discovered that emerin binds directly to several transcription repressors, including GCL. Interestingly, BAF competes with GCL for binding to emerin in vitro, suggesting that BAF might antagonize repressor binding to emerin in vivo. We further discovered that BAF binds directly to histone H 1 and histone H3, suggesting that BAF interacts with nucleosomes and may either regulate or be influenced by chromatin structure in vivo. We propose that emerin and BAF form a variety of protein complexes at the inner nuclear membrane, which affect chromatin structure and gene regulation. We discovered a mammalian protein named BFL, which is 54% identical to BAF. BFL interacts with BAF and LAP2beta, but does not bind to DNA or emerin. BFL mRNA is expressed in most tissues, but its absence from heart and skeletal muscle (selectively affected by EDMD disease) suggests that BFL might protect other tissues from the loss of emerin. Our models for the mechanisms and functions of BAF, BFL and emerin will be tested using biochemistry, microscopy, cell-free Xenopus extracts, and in vivo analysis in human cell lines. We have four specific aims. (1) Determine which domains in histone H1 and H3 bind BAF; test the hypothesis that BAF interacts with nucleosomes or influences HI-mediate chromatin compaction; determine if BAF binding to histone H3 is regulated by posttranslational modifications of the H3 tail. (2) Determine how phosphorylation affects BAF binding to emerin and other partners, and dissect structural roles for BAF during nuclear assembly in vitro. (3) Test the hypothesis that emerin represses gene expression, using luciferase reporter assays in cultured mammalian cells; we will also use our large collection of biochemically-characterized mutations in emerin and BAF to understand the functions of different emerin complexes both in vitro and in living cells. (4) Use a proven transcriptional repressor, LAP2beta, in luciferase reporter assays to test our hypothesis that BFL or BAF regulate LAP2beta repression in vivo; use biochemical competition assays to determine which LAP2beta- or emerin-binding proteins compete, or cooperate, with each other for binding to LAP2beta or emerin. Our goal is determine which partners contribute to repressive LEM-domain protein complexes at the inner nuclear membrane, and which partners anchor these complexes.