Studying chromosome function using chemical biology

描述（由申请人提供）：DNA被包裹在由组蛋白组成的线轴上，这一事实被认为基本上影响了染色体生物学的各个方面，包括DNA复制、损伤后修复和分离。组蛋白的多种翻译后修饰（如乙酰化、甲基化和磷酸化）是已知的，并且被认为在调节与我们的基因组相关的广泛生物学方面发挥着关键作用。根据观察到的不同组蛋白翻译后修饰（或“标记”）在向染色体招募蛋白质时的拮抗和协同作用，已经提出这些“标记”形成了调节染色体功能的“密码”。也有人提出，这种“密码”可能为表观遗传提供了基础，表观遗传是细胞特征的传递，而不是在DNA序列水平上编码。许多翻译后修饰组蛋白的蛋白质(如：“写”或“擦除”（“代码”）的特征。相比之下，我们对识别（或“读取”）组蛋白翻译后修饰的蛋白质的知识仍然不完整。识别这些效应蛋白（或“读取器”）的困难在很大程度上是由于组蛋白修饰是亚化学计量的、动态的和弱相互作用的介质。为了填补这一知识空白，我们最近报道了一种将光化学交联与生物正交化学相结合的方法，以“捕获”与Lys-4三甲基化的组蛋白H3结合的蛋白质。我们现在将这种方法与最先进的质谱法相结合，开发出一种强大的化学蛋白质组学方法来分析组蛋白甲基化标记的“读者”。我们正在进行的工作表明，我们的方法是通用的，可用于分析任何人类细胞类型（如正常或癌症）、细胞状态（如有丝分裂）或环境（如药物治疗）中这些翻译后修饰依赖的蛋白质-蛋白质相互作用。基于这些和其他未发表的初步数据，我们建议：(i)全面分析识别组蛋白上甲基化“标记”的蛋白质，（ii）表征识别甲基化“标记”的蛋白质如何控制下游生物学，以及（iii）研究组蛋白磷酸化和甲基化之间的相互作用如何确保细胞分裂过程中无错误的染色体分离。我们结合化学，生物化学，高分辨率显微镜和细胞生物学方法来深入了解基本的细胞过程。我们的研究结果可能揭示了组蛋白翻译后修饰的不当“阅读”如何导致疾病。从长远来看，我们的发现也可能为开发针对组蛋白甲基化“标记”的“读者”的新治疗策略提供基础。

DESCRIPTION (provided by applicant): The fact that DNA is wrapped around a spool, comprised of histones, is believed to influence essentially all aspects of chromosome biology, including DNA replication, repair after damage and segregation. Diverse post-translational modifications (e.g. acetylation, methylation, and phosphorylation) of histones are known and are believed to play key roles in regulating a wide swath of biology linked to our genomes. Based on observed antagonisms and synergies between different histone post-translational modifications (or 'marks') in recruiting proteins to chromosomes, it has been proposed that these 'marks' form a 'code' for regulating chromosome function. It has also been suggested that this 'code' may provide a basis of epigenetic inheritance, which is the transmission of cellular traits that are not encoded at the level of DNA sequence. Many of the proteins that post-translationally modify histones (i.e. 'write' or 'erase' the 'code') have been characterized. In contrast, our knowledge of the proteins that recognize (or 'read') histone post-translational modifications remains incomplete. The difficulty in identifying these effector-proteins (or 'readers') is, in large part, due to the histone modifications being sub-stoichiometric, dynamic, and mediators of weak interactions. With the goal to fill this knowledge gap, we have recently reported an approach, which combines photo-chemical crosslinking with bio-orthogonal chemistry, to 'capture' proteins that bind histone H3 trimethylated at Lys-4. We now combine this method with state-of-the-art mass spectrometry to develop a robust chemical proteomics approach to profile 'readers' of histone methylation 'marks.' Our ongoing work suggests that our approach is general and can be used to analyze these post-translational modification-dependent protein-protein interactions in any human cell type (e.g. normal or cancer), cell state (e.g. mitosis) or context (e.g. drug-treated). Based on these and other unpublished preliminary data, we propose to: (i) comprehensively profile proteins that recognize methylation 'marks' on histones, (ii) characterize how proteins that recognize methylation 'marks' control down-stream biology, and (iii) examine how interplay between histone phosphorylation and methylation ensures error-free chromosome segregation during cell division. We combine chemistry, biochemistry, high-resolution microscopy and cell biological approaches to gain insight into fundamental cellular processes. Our findings may reveal how improper 'reading' of histone post-translational modifications can result in disease. In the long-term, our findings may also provide a basis for developing new therapeutic strategies that target 'readers' of histone methylation 'marks'. PUBLIC HEALTH RELEVANCE: The goals of the proposed research are to develop and apply a chemical proteomics approach to comprehensively profile proteins that recognize histones with particular post-translational modifications (or 'marks'). By characterizing these proteins, we will gain insight into how chromosome function is regulated in normal cells and how diseases may arise when the 'reading' of these histone 'marks' goes awry.