Studying chromosome function using chemical biology

 描述（由申请人提供）：细胞分裂中的错误，即复制的DNA在两个子细胞之间分配的过程，与疾病和发育缺陷有关。不适当的细胞分裂也被广泛用于治疗疾病，如癌症的治疗策略中。准确的染色体分离、适当的细胞周期进程和DNA损伤的修复依赖于识别组蛋白上的翻译后修饰的关键蛋白质，组蛋白组装被称为核小体的染色质的基本单位。使用常规方法来全面分析“读取”组蛋白上的特定翻译后修饰（或“标记”）以调节染色体生物学的蛋白质是困难的。我们的目标是通过设计新的基于化学的策略来填补这一知识空白，以在特定的细胞环境中描述组蛋白“标记”的直接“读者”。我们的方法的核心是通过共价键，使用光交联剂将动态和弱（通常为微摩尔）蛋白质-蛋白质相互作用转化为稳定的缔合。这种策略与最先进的定量质谱法相结合，以确定特定组蛋白“标记”的“读者”。在完成项目期间，我们开发了这种“化学蛋白质组学”方法，命名为CLASPI（交联辅助和基于SILAC的蛋白质鉴定），并证明了其识别组蛋白“标记”的新“读者”的能力，包括组蛋白H3甲基化和磷酸化。我们将建立在我们最近的出版物和初步数据，并将重点确定两个不同的组蛋白磷酸化“标记”的“读者”，一个表示DNA损伤，并在细胞毒性药物的长期有丝分裂停滞期间观察到，另一个与分裂细胞中的染色体相关，并取决于极光激酶的活性，极光激酶是一种保守的细胞分裂调节因子，也是抗癌药物的靶点。在细胞周期的不同阶段“阅读”这些组蛋白“标记”的功能意义将使用高分辨率显微镜分析、敲除关键蛋白的小鼠胚胎成纤维细胞和在快速时间尺度上作用以阻断组蛋白“标记”活性的化学抑制剂来检查。 负责在细胞中产生这些“标记”的激酶。该提案有三个目标：（i）鉴定“读取”组蛋白H2 AX上磷酸化“标记”的蛋白质，（ii）表征组蛋白H2 AX磷酸化-“读取器”的功能，和（iii）分析活细胞中组蛋白翻译后修饰的“读取器”。拟议的研究结合了化学和生物学方法，以揭示组蛋白“标记”如何被蛋白质“解释”，以通过调节染色体分离，DNA损伤修复和细胞周期进程来确保稳定的基因组繁殖。这些研究还应该阐明细胞分裂的化学抑制剂如何杀死癌细胞。此外，关键的翻译后修饰依赖的蛋白质-蛋白质相互作用的综合分析应导致治疗剂的新靶点的选择。最后，我们开发的方法是通用的，可以广泛应用于解剖细胞中蛋白质-蛋白质相互作用的复杂和动态网络。

 DESCRIPTION (provided by applicant): Errors in cell division, the process during which replicated DNA is partitioned between two daughter cells, have been linked to diseases and developmental defects. Improper cell division has also been exploited in therapeutic strategies widely used to treat diseases, such as cancer. Accurate chromosome segregation, proper cell cycle progression and repair of DNA damage rely on key proteins recognizing post-translational modifications on histones, which assemble the basic units of chromatin known as nucleosomes. Comprehensively profiling proteins that `read' specific post-translational modifications (or `marks') on histones to regulate chromosome biology has been difficult using conventional approaches. Our goal is to fill this knowledge gap by devising new chemistry-based strategies to profile direct `readers' of histone `marks' in specific cellular contexts. Central to our approach s the conversion of dynamic and weak (typically micromolar) protein-protein interactions into stable associations, via covalent bonds, using photo-cross-linkers. This strategy is combined with state-of-the-art quantitative mass spectrometry to identify `readers' of specific histone `marks'. In the completed project period, we have developed this `chemical proteomics' approach, named CLASPI (cross-linking-assisted and SILAC-based protein identification), and demonstrated its ability to identify new `readers' of histone `marks', including histone H3 methylation and phosphorylation. We will build on our recent publications and preliminary data and will focus on identifying `readers' of two different histone phosphorylation `marks', one that indicates DNA damage and is observed during prolonged mitotic arrest with cytotoxic drugs, and another that associates with chromosomes in dividing cells and depends on the activity of Aurora kinase, a conserved regulator of cell division and a target of anti-cancer drugs. The functional significance of `reading' these histone `marks' at different stages of the cell cycle wil be examined using high-resolution microscopy assays, mouse embryonic fibroblasts with key proteins knocked out, and chemical inhibitors that act on fast time-scales to block the activity of kinases responsible for generating these `marks' in cells. The proposal has three aims: (i) To identify proteins that `read' a phosphorylation `mark' on histone H2AX, (ii) To characterize the functions of histone H2AX phosphorylation-`readers', and (iii) To profile `readers' of histone post-translational modifications in living cells. The proposed research combines chemistry and biology approaches to unravel how histone `marks' are `interpreted' by proteins to ensure stable genome propagation by regulating chromosome segregation, DNA damage repair and cell cycle progression. These studies should also shed light on how chemical inhibitors of cell division kill cancer cells. In addition, the comprehensive profiling of key post-translational modification-dependent protein-protein interactions should lead to the selection of new targets for therapeutic agents. Finally, the approaches we develop are general and can be broadly applied to dissect complex and dynamic networks of protein-protein interactions in cells.