基于多价亲和标记探针的组蛋白修饰阅读器分离分析新方法

The specific recognition of histone modification by readers recruits various components of the nuclear signaling network to mediate fundamental process such as gene transcription and its downstream pathways. Misreading of the important epigenetic markers has been shown to underlie a host of human disease, including cancer. Thus, its essential to comprehensively profile readers that recognize histone marks. However, the noncovalent interactions between histone modification and readers are rather weak, transient, and are highly dynamic, the separation and analysis of histone readers remain a great challenge. The present research proposal aims at developing new affinity labeling probes based on multivalent organocatalysts. Through the proximity effect and acyl transfer reaction mediated by “peptide-target” specific interaction, the probes are able to selectively label their target proteins. Then, target proteins are separated and enriched based on their biotin-labels for subsequent identification and quantification analysis by LC-MS. Finally, the MS proteomic data will be used to profile the potential readers of histone crotonylation and 3-Hydroxybutyrylation. Isothermal titration calorimetry measurement and molecular docking analysis will also be applied to verify the newly identified readers towards the histone modifications. This proposed project would not only establish a new method for the separation and analysis of histone modification readers, but also provide novel insights for the epigenetic research.

组蛋白修饰阅读器蛋白通过识别特定组蛋白修饰，调控基因表达及下游通路，其功能紊乱与肿瘤等疾病发生密切联系，因此组蛋白修饰阅读器的分离分析具有重要的生物学意义，是当前国际研究前沿课题。然而，组蛋白修饰与其阅读器结合力极弱，且具有瞬间、动态变化等特点，目前尚缺乏高特异性的分析方法，因此阅读器蛋白的分离分析具有极大的挑战性。本研究拟发展基于有机小分子催化的多价亲和标记多肽探针，以组蛋白甲基化修饰为模型，通过“多肽-蛋白”特异识别介导的邻近效应与酰基转移反应，实现对目标蛋白的选择性标记；继而，基于生物素酰化标签分离、富集阅读器蛋白，应用液相色谱-质谱联用技术进行定性和定量分析，鉴定组蛋白巴豆酰化等新修饰的潜在阅读器蛋白；进而采用等温滴定量热法、分子对接分析等手段对新鉴定的阅读器进行验证和分子识别机制研究。本项目研究有望在建立一个组蛋白修饰阅读器分离分析新方法的同时，为表观遗传学研究提供新视角。

组蛋白修饰阅读器（结合蛋白）通过识别特定组蛋白修饰调控基因表达而发挥生物功能；因此，组蛋白修饰阅读器的分析鉴定具有重要的生物学意义。然而，组蛋白修饰与其阅读器结合力极弱，且具有瞬间、动态变化等特点，因此阅读器蛋白的分离分析具有极大的挑战性。本项目基于遗传密码拓展系统、邻近标记和定量蛋白质组学策略，发展了一种分析细胞内组蛋白特定修饰位点阅读器的新方法；项目以组蛋白巴豆酰化修饰为模型，将巴豆酰赖氨酸（Kcr）位点特异的插入组蛋白H3而表达融合蛋白APEX2-H3K9cr；进而激活APEX2对H3K9cr结合蛋白进行高效生物素化标记；继而，分离富集生物素化蛋白，应用定量蛋白质组学鉴定该修饰位点结合蛋白；进一步通过生物信息学分析发现DPF2为其潜在阅读器，并采用等温滴定量热法、分子对接分析等手段证实了DPF2-H3K9cr的高特异识别。本项目研究为组蛋白修饰结合蛋白的分析鉴定提供了一个新方法，同时，也为表观遗传学研究提供了新视角。