Li Guofang, Meng Fanxu, Zhong Xiaomin, Yu Kairan, Zhang Nana, Zhang Keren, Huang Huang, Li Wenli, Zhang Jianing, Wang Wei, Ren Yan, Liu Yubo
Cancer Hospital of Dalian University of Technology, Liaoning Cancer Hospital & Institute, Shenyang, China.
Department of Life and Pharmaceutical Sciences, Dalian University of Technology, Panjin, China.
Sci Rep. 2025 Jul 2;15(1):22679. doi: 10.1038/s41598-025-07074-7.
O-linked β-N-acetylglucosamine (O-GlcNAc), a critical post-translational modification predominantly found in the nucleus, plays a substantial role in regulating gene expression by modulating transcription factors (TFs) activity. However, quantitative analysis investigating the influence of O-GlcNAcylation on protein-DNA interactions at a proteome scale remains undone. Herein, a pulldown screening approach using a consensus TF response element (catTFRE) was employed to unravel the impact of fluctuating levels of O-GlcNAcylation on the DNA binding efficiency of endogenous TFs/co-factors. Utilizing quantitative proteomics, we identified a substantial enhancement in the binding capacity of 241 nuclear proteins (NPs) to DNA sequences due to elevated levels of O-GlcNAcylation, whereas a decrease in DNA binding was observed for 2 NPs concurrently. Intriguingly, the O-GlcNAcylation elevation significantly enhanced the binding of 146 TFs/co-factors to specific DNA sequences. We further established that the O-GlcNAcylation of several Forkhead family TFs, including FOXA1 and FOXC1, notably enhances their binding to specific DNA sequences in living cells. Our research presents an efficacious approach to assessing the impact of O-GlcNAcylation on the interactions between proteins and DNA. This significantly enhances our understanding of the role O-GlcNAcylation plays in the regulation of transcription.
O-连接的β-N-乙酰葡糖胺（O-GlcNAc）是一种主要存在于细胞核中的关键翻译后修饰，通过调节转录因子（TFs）的活性在基因表达调控中发挥重要作用。然而，在蛋白质组规模上研究O-GlcNAcylation对蛋白质-DNA相互作用影响的定量分析尚未完成。在此，我们采用了一种使用共有TF反应元件（catTFRE）的下拉筛选方法，以揭示O-GlcNAcylation水平波动对内源TFs/辅因子与DNA结合效率的影响。利用定量蛋白质组学，我们发现由于O-GlcNAcylation水平升高，241种核蛋白（NPs）与DNA序列的结合能力显著增强，而同时观察到2种NPs与DNA的结合减少。有趣的是，O-GlcNAcylation升高显著增强了146种TFs/辅因子与特定DNA序列的结合。我们进一步证实，包括FOXA1和FOXC1在内的几种叉头家族TFs的O-GlcNAcylation显著增强了它们在活细胞中与特定DNA序列的结合。我们的研究提出了一种评估O-GlcNAcylation对蛋白质与DNA相互作用影响的有效方法。这显著增强了我们对O-GlcNAcylation在转录调控中所起作用的理解。
