VPS72/YL1 function in nuclear re-assembly

VPS72/YL1 在核重组中的功能

• 批准号: 515940343
• 负责人: Professor Dr. Wolfram Antonin
• 金额: --
• 依托单位: Institut für Biochemie und Molekulare Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/515940343?language=en
• 关键词: VPS72; YL1; function; nuclear; re

The nucleus reorganizes structurally and functionally considerably during mitosis. In animal cells, the nuclear envelope breaks down at the beginning of mitosis, the chromatin massively condenses to individualized chromosomes, which are captured and segregated by the spindle apparatus - processes that have been and still are intensively studied in many laboratories. We know much less how at the end of mitosis the interphase state of the nucleus is reestablished, competent for its manifold functions. For this, the highly condensed mitotic chromosomes are decompacted. Around the decondensing chromatin the nuclear envelope re-assembles, nuclear substructures like nucleoli reform and nuclear transport re-establishes compartmentalization. These processes are indispensable for reinitiating transcription and the perpetuation of genomic information and thus of central importance in the cellular life cycle. Despite its significance to basic research as well as its potential medical implications, nuclear reformation during mitotic exit is in molecular terms still ill defined. We have identified VPS72/YL1, a chaperone of the histone variant H2A.Z, and its conserved YL-1 domain as a crucial factor for nuclear re-assembly: In Xenopus egg extracts, where nuclear reformation can be conveniently reconstituted, depletion of VPS72 blocks H2A.Z integration into chromatin and leads to nuclear structure defects. The chromatin appears disorganized and the nuclear envelope is irregular and reaches into areas usually occupied by chromatin. The YL-1 domain, in turn, is not required for bulk H2A.Z chromatin loading but has a specific, yet unknown, function in nuclear re-assembly. Interestingly, this domain interacts with the spindle assembly checkpoint components MAD2 and p31 and a less defined factor, WDR83. Depletion of p31 and MAD2 compromises the nuclear structure as VPS72 depletion indicating a non-canonical function of the checkpoint proteins p31 and MAD2. Here, we will define the function of the YL-1 domain and its binding partners MAD2, p31 and WDR83 in nuclear re-assembly using Xenopus egg extracts and cellular assays. The second includes CRISPR/CAS based knockouts or inducible degradation of target proteins in tissue culture cells combined with life cell imaging. We will characterize the function of the VPS72/MAD2/p31/WDR83 interaction network for nuclear assembly and define which aspects of nuclear reformation are compromised if these components are missing. We will test the hypothesis that the YL-1 domain is required to load H2A.Z to or recognize specific chromatin loci, which in turn are crucial for proper nuclear re-assembly. For this, we use CHIP experiments in Xenopus egg extracts, to be established in this system, and in tissue culture cells. By a combination of cell-free and cellular assays, we will shed light on an ill-defined but important cell biological process at the end of mitosis vital to reestablish nuclear structure and function.

在有丝分裂过程中，细胞核在结构和功能上进行了相当大的重组。在动物细胞中，核膜在有丝分裂开始时破裂，染色质大量凝聚成个体化的染色体，这些染色体被纺锤体捕获并分离-许多实验室已经并仍然在深入研究这一过程。在有丝分裂结束时，细胞核的间期状态是如何重建的，如何胜任它的多种功能，我们所知道的就少得多了。为此，高度浓缩的有丝分裂染色体被解压缩。在去致密染色质周围，核膜重新组装，核亚结构如核仁改革和核运输重新建立区室化。这些过程对于重新启动转录和基因组信息的永久化是必不可少的，因此在细胞生命周期中至关重要。尽管其重要性的基础研究，以及其潜在的医学意义，核重组在有丝分裂退出是在分子方面仍然不明确。我们已经鉴定了VPS 72/YL 1，组蛋白变体H2A.Z的伴侣蛋白，及其保守的YL-1结构域作为核重组的关键因素：在爪蟾卵提取物中，核重组可以方便地重建，VPS 72的耗尽阻断了H2A.Z整合到染色质中并导致核结构缺陷。染色质排列紊乱，核被膜不规则，伸入染色质常占据的区域。反过来，YL-1结构域不是批量H2A.Z染色质加载所需的，但在核重组中具有特异性但未知的功能。有趣的是，该结构域与纺锤体组装检查点组件MAD 2和p31以及定义较少的因子WDR 83相互作用。p31和MAD 2的消耗损害了核结构，因为VPS 72消耗指示检查点蛋白p31和MAD 2的非典型功能。在这里，我们将使用非洲爪蟾卵提取物和细胞测定来定义YL-1结构域及其结合伙伴MAD 2、p31和WDR 83在核重组中的功能。第二种包括基于CRISPR/CAS的敲除或组织培养细胞中靶蛋白的诱导性降解，结合生命细胞成像。我们将描述VPS 72/MAD 2/p31/WDR 83相互作用网络在核组装中的功能，并定义如果这些组件缺失，核重组的哪些方面会受到影响。我们将检验YL-1结构域是将H2A.Z加载到或识别特定染色质基因座所需的假设，这反过来对正确的核重组至关重要。为此，我们在爪蟾卵提取物中使用CHIP实验，在该系统中建立，并在组织培养细胞中。通过无细胞和细胞分析的结合，我们将阐明在有丝分裂结束时对重建核结构和功能至关重要的一个不明确但重要的细胞生物学过程。