基于代谢标记与多组学分析发现O-GlcNAc糖基化修饰的转录因子及其在人乳腺癌细胞耐药中的作用

O-GlcNAcylation defines as O-linked-N-acetylglucosamine (O-GlcNAc) on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins, which plays crucial regulatory roles in almost all physiological and pathological processes. Our preliminary results suggest the multidrug resistance of human breast cancer cells is modulated via O-GlcNAcylation of transcription factors, but the detailed mechanism remains unclear. This study aims to characterize the O-GlcNAcylated transcription factor and its roles in multidrug resistance of human breast cancer cells. 1) Using feeding metabolic labeling combined with ChIP-Seq like method, O-GlcNAc metabolic labeling Chromatin-Affinity Precipitation DNA Sequencing (O-GlcNAc-ChAP-Seq) analysis strategy will be established firstly. By integrating O-GlcNAc-ChAP-Seq with proteomics analyses and next-generation DNA sequencing, O-GlcNAcylated transcription factors will be profiled and their whole genomic targeted genes will be mapped in human breast cancer cells with or without multidrug resistance. Together with transcriptomics RNA-seq analyses further, the O-GlcNAcylation regulated multidrug resistant gene expression network model will be approached. 2) Some novel O-GlcNAcylated transcription factors related to multidrug resistance will be identified. Their function and potential O-GlcNAcylation sites will be characterized. 3) The role of O-GlcNAcylated transcription factors, such as ATF3 and RREB1, will be elucidated in the regulation of multidrug resistant-related signaling pathways such as cell stress response, DNA damage repair and apoptosis. 4) To reverse the drug resistance of breast cancer cells in vitro, the abnormal O-GlcNAcylation will be intervened. This study will contribute to develop the mechanism of O-GlcNAcylation involved in the regulation of drug resistance-related gene expression network in breast cancer comprehensively and systematically, and provide a new treatment strategy for reversal of drug resistance in breast cancer as well.

O-GlcNAc糖基化是细胞内的一种重要单糖修饰，参与多种生理和病理过程调控。本研究将基于O-GlcNAc糖代谢标技术，1）发展O-GlcNAc糖代谢标记转录因子组结合的染色质DNA序列分析方法，联合O-GlcNAc糖代谢标记转录因子蛋白组学和转录组学手段，实现针对化疗药物耐药与敏感人乳腺癌细胞的多组学贯通比较分析，提出O-GlcNAc修饰调控的耐药相关基因表达网络模型；2）发现新的乳腺癌耐药相关O-GlcNAc糖基化修饰转录因子，分析其O-GlcNAc修饰位点和功能；3）阐释ATF3、RREB1等O-GlcNAc糖基化转录因子在调控细胞应激、DNA损伤修复、凋亡等耐药相关信号通路的作用；4）通过干预和调控异常的O-GlcNAc糖基化修饰，在体外实现逆转乳腺癌细胞耐药性。本研究旨在全面、系统揭示O-GlcNAc糖基化参与乳腺癌耐药相关基因表达网络调控的机制，为乳腺癌耐药逆转治疗提供新思路。

O-GlcNAc糖基化是细胞内的一种重要单糖修饰，参与多种生理和病理过程调控。研究表明，O-GlcNAc糖基化在调节药物刺激等应激反应和细胞稳态中发挥重要作用。本研究发展了一种基于代谢标记的化学选择性O-GlcNAc染色质测序（chemoselective O-GlcNAc chromatin sequencing，COGC-seq）方法，以此为工具，结合代谢标记的O-GlcNAc染色质结合蛋白定量蛋白质组学和转录组学技术，从多组学角度系统分析了O-GlcNAc糖基化的转录调控因子及其靶基因在乳腺癌细胞基因毒（阿霉素）应激反应机制中的作用。与亲代乳腺癌细胞MCF-7相比，阿霉素耐药的乳腺癌细胞MCF-7/ADR的染色质O-GlcNAc修饰显著上调，提示这种糖基化对基因毒应激反应的转录重编程存在调控作用。定量蛋白质谱分析获得了875个差异O-GlcNAc染色质结合蛋白（OCPs），包括88个O-GlcNAc修饰的转录因子或辅因子（OCTFs）。利用COGC-seq，该研究进一步将OCPs在染色质上的结合位置和强度进行分析。结果表明，基因毒刺激在全基因组范围内驱使OCPs结合位置从启动子-增强子平衡态转换为启动子偏向态。随着O-GlcNAc修饰上调，OCPs在染色质上结合强度增强，并进一步激活下游基因的转录。通过多组学整合分析，该研究建立了基因毒诱导的OCTFs调控下游靶基因表达的网络模型。其中，O-GlcNAc修饰的NRF1在该转录调控网络中扮演关键角色。O-GlcNAc糖基化激活了该转录因子的染色质结合能力和转录调控功能，这对MCF-7/ADR的基因毒耐受表型起到重要影响。这些结果揭示了O-GlcNAc糖基化可以作为应激传感器调控多种基因表达，细胞以此应答外界刺激并维持稳态，在乳腺癌等肿瘤细胞耐药过程中发挥重要作用。

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