Regulation of nuclear envelope assembly and disassembly

长期目标是了解核膜和层蛋白细丝是如何支持基因的 原子核的活动，这是生物学的一个开放前沿。我们将重点介绍障碍到自整合系数 (Baf)，一种DNA结合蛋白，在多细胞动物中是保守的和必不可少的。巴夫有钥匙 核组装过程中涉及其主要伙伴的结构作用：染色质、层粘连蛋白和 丰富的LEM结构域核蛋白家族，包括核膜蛋白Emerin。 Emerin或A型板层的突变会导致Emery-Dreifuss肌营养不良症。BAF还拥有 肌肉中的作用：线虫需要维持成体肌肉，并对其过度表达产生扰动 培养的小鼠成肌细胞的分化。人们对曝气生物滤池最不了解的方面是它的相互作用 染色质，特别是在体细胞中。BAF调节高度组织特异性的基因表达 举止。BAF直接与核心组蛋白H3和H4结合，提示它与核小体相互作用， 染色质结构的基本单位。核小体是许多调节更高秩序的因素的靶标。 折叠和转录潜力(活跃与沉默染色质)。组蛋白H3和H4 在过度表达BAF的细胞中，乙酰化减少，在BAF缺乏的细胞中，乙酰化增加，提示 BaF可能抑制组蛋白乙酰化。此外，重组BAF通过以下途径抑制组蛋白H3的乙酰化 纯化的Gcn5，一种组蛋白乙酰转移酶(HAT)，表明BAF可能阻止酶进入 组蛋白。BAF是否影响其他组蛋白翻译后修饰尚不清楚。我们 假设BAF是一种新的表观遗传调节因子。我们将测试两种非独占机制 从而影响染色质。在核小体组织者模型中，BAF二聚体是 建议通过与DNA、H3、H4或其组合结合来致密核小体。这 压缩可能是BAF在组织核染色质方面的一种通用功能，具有潜在的 立体阻断染色质修饰因子。在系列式生物滤池模型中，提出了以生物滤池为目标 通过直接与特定转录因子结合的特定启动子。一旦被拴住，BAF可能会被阻挡 组蛋白乙酰化(或其他修饰)，或可能招募替代染色质修饰 各种因素。目标1将通过对BAF的结构分析来检验BAF致密核小体的假设 BAF与单核小体、12聚体核小体阵列或12聚体形成的产物 核小体阵列加上连接子组蛋白。目标2将确定其他表观遗传修饰，由 Baf在细胞中的表达，并鉴定亲和蛋白纯化的新转录因子和染色质调节因子 和BAF在一起。目的3研究细菌滤泡因子功能的全基因组和启动子特异性分析。 将测试BAF-1突变体对Baf-1缺失表型的功能挽救及其对 组蛋白修饰在特定的BAF-1调节启动子。

The long-term goals are to understand how the nuclear envelope and lamin filaments support gene activity in the nucleus, an open frontier in biology. We will focus on Barrier-to-Autointegration Factor (BAF), a DNA-binding protein that is conserved and essential in multicellular animals. BAF has key structural roles during nuclear assembly that involve its major partners: chromatin, lamins and the abundant ¿LEM-domain¿ family of nuclear proteins including emerin, a nuclear membrane protein. Mutations in either emerin or A-type lamins cause Emery-Dreifuss muscular dystrophy. BAF also has roles in muscle: it is required to maintain adult muscles in C. elegans, and its over-expression perturbs differentiation of cultured mouse myoblasts. The least-understood aspects of BAF are its interactions with chromatin, particularly in somatic cells. BAF regulates gene expression in a highly tissue-specific manner. BAF binds directly to core histones H3 and H4, suggesting it interacts with nucleosomes, the basic unit of chromatin structure. Nucleosomes are targeted by numerous factors that regulate higherorder folding and transcriptional potential (¿active¿ vs ¿silenced¿ chromatin). Histone H3 and H4 acetylation is reduced in cells that overexpress BAF, and increases in BAF-deficient cells, suggesting BAF might inhibit histone acetylation. Furthermore recombinant BAF inhibits histone H3 acetylation by purified Gcn5, a histone acetyltransferase (HAT), suggesting BAF might block enzyme access to histones. Whether BAF influences other histone posttranslational modifications is unknown. We hypothesize that BAF is a novel epigenetic regulator. We will test two nonexclusive mechanisms by which BAF might influence chromatin. In the ¿nucleosome organizer¿ model, BAF dimers are proposed to compact nucleosomes through binding to DNA, H3, H4 or combinations thereof. This compaction might be a ¿generic¿ function for BAF in organizing nuclear chromatin, with the potential to sterically block chromatin-modifying factors. In the ¿tethered BAF¿ model, BAF is proposed to target specific promoters by direct binding to specific transcription factors. Once tethered, BAF might block histone acetylation (or other modifications) sterically, or might recruit alternative chromatin-modifying factors. Aim 1 will test the hypothesis that BAF compacts nucleosomes, by structural analysis of the products formed by BAF plus either mononucleosomes, 12-mer arrays of nucleosomes, or 12-mer nucleosome arrays plus linker histones. Aim 2 will identify other epigenetic modifications regulated by BAF in cells, and characterize novel transcription factors and chromatin regulatory factors that affinitypurify with BAF. Aim 3 is the whole-genome and promoter-specific analysis of BAF function in C. elegans; BAF-1 mutants will be tested for functional rescue of baf-1 null phenotypes and their affects on histone modifications at a specific BAF-1-regulated promoter.