DNA甲基化是通过DNA甲基转移酶(Dnmts)在整个基因组中建立和维持的关键表观遗传修饰。DNA甲基化模式的改变在许多肿瘤基因组中经常发生,和Dnmts的抑制剂已成为重要的表观遗传药物。阿扎胞苷是一种胞苷类似物,可掺入DNA并诱导Dnmts的特异性抑制和蛋白酶体介导的降解。阿扎胞苷对CpG甲基化和基因转录的下游效应已在许多系统中被广泛研究。但阿扎胞苷对蛋白质组的影响尚不清楚。此外,具有诱导Dnmts快速降解的特定能力(特别是,主要维持DNA甲基转移酶,Dnmt1),它可以用作研究Dnmt1相关的功能或物理相互作用的特异性化学敲除。在这项研究中,我们使用定量蛋白质组学分析阿扎胞苷处理的细胞核蛋白质组中蛋白质的降解谱.我们确定了特定的蛋白质以及受阿扎胞苷影响的多个途径和过程。Dnmt1交互伙伴,Uhrf1,表现出显着阿扎胞苷诱导的降解,这种阿扎胞苷诱导的降解与Dnmt1蛋白的水平无关。我们确定了多种其他与色素和表观遗传相关的因素,包括含溴结构域的转录调节因子,Brd2.我们表明阿扎胞苷诱导Dnmt1相关蛋白质组的高度特异性扰动,虽然互动伙伴如Uhrf1对阿扎胞苷敏感,其他如Dnmt1相互作用伙伴和稳定性调节剂,Usp7不是。总之,我们对阿扎胞苷敏感的核蛋白质组进行了首次全面的蛋白质组学分析,我们展示了5-阿扎胞苷如何用作探索Dnmt和染色质相关蛋白质网络的特异性探针。
DNA methylation is a critical epigenetic modification that is established and maintained across the genome by DNA methyltransferase enzymes (Dnmts). Altered patterns of DNA methylation are a frequent occurrence in many tumor genomes, and inhibitors of Dnmts have become important epigenetic drugs. Azacitidine is a cytidine analog that is incorporated into DNA and induces the specific inhibition and proteasomal-mediated degradation of Dnmts. The downstream effects of azacitidine on CpG methylation and on gene transcription have been widely studied in many systems, but how azacitidine impacts the proteome is not well-understood. In addition, with its specific ability to induce the rapid degradation of Dnmts (in particular, the primary maintenance DNA methyltransferase, Dnmt1), it may be employed as a specific chemical knockdown for investigating the Dnmt1-associated functional or physical interactome. In this study, we use quantitative proteomics to analyze the degradation profile of proteins in the nuclear proteome of cells treated with azacitidine. We identify specific proteins as well as multiple pathways and processes that are impacted by azacitidine. The Dnmt1 interaction partner, Uhrf1, exhibits significant azacitidine-induced degradation, and this azacitidine-induced degradation is independent of the levels of Dnmt1 protein. We identify multiple other chromatin- and epigenetic-associated factors, including the bromodomain-containing transcriptional regulator, Brd2. We show that azacitidine induces highly specific perturbations of the Dnmt1-associated proteome, and while interaction partners such as Uhrf1 are sensitive to azacitidine, others such as the Dnmt1 interaction partner and stability regulator, Usp7, are not. In summary, we have conducted the first comprehensive proteomic analysis of the azacitidine-sensitive nuclear proteome, and we show how 5-azacitidine can be used as a specific probe to explore Dnmt- and chromatin-related protein networks.