Studying chromosome function using chemical biology

描述（由申请人提供）：DNA缠绕在由组蛋白组成的线轴上的事实被认为基本上影响染色体生物学的所有方面，包括DNA复制、损伤后的修复和分离。组蛋白的多种翻译后修饰（例如乙酰化、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关键作用。基于观察到的不同组蛋白翻译后修饰（或“标记”）在将蛋白质募集到染色体中时之间的拮抗和协同作用，已经提出这些“标记”形成用于调节染色体功能的“代码”。也有人认为，这种“密码”可能提供了表观遗传的基础，表观遗传是指在DNA序列水平上没有编码的细胞特征的传递。许多蛋白质的后修饰组蛋白（即'写'或'擦除'的'代码'）已被确定。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了解仍然不完整。识别这些效应蛋白（或“阅读器”）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方法，该方法将光化学交联与生物正交化学相结合，以“捕获”结合在Lys-4处三甲基化的组蛋白H3的蛋白。我们现在联合收割机将这种方法与最先进的质谱技术相结合，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（例如正常或癌症），细胞状态（例如有丝分裂）或背景（例如药物治疗）中的这些翻译后修饰依赖性蛋白质-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。我们结合联合收割机化学，生物化学，高分辨率显微镜和细胞生物学方法，以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”是如何导致疾病的。从长远来看，我们的研究结果也可能为开发新的治疗策略提供基础，这些策略针对组蛋白甲基化“标记”的“读者”。 公共卫生相关性：拟议研究的目标是开发和应用化学蛋白质组学方法来全面分析识别具有特定翻译后修饰（或“标记”）的组蛋白的蛋白质。通过表征这些蛋白质，我们将深入了解染色体功能在正常细胞中是如何调节的，以及当这些组蛋白“标记”的“阅读”出错时，疾病是如何发生的。

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.