Proteomic Analysis of Azacitidine-Induced Degradation Profiles Identifies Multiple Chromatin and Epigenetic Regulators Including Uhrf1 and Dnmt1 as Sensitive to Azacitidine

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.

DNA甲基化（DNA methylation）是一类关键的表观遗传修饰，由DNA甲基转移酶（DNA methyltransferases, Dnmts）在全基因组范围内建立并维持。DNA甲基化模式异常在众多肿瘤基因组中频繁出现，而Dnmts抑制剂已成为重要的表观遗传药物。阿扎胞苷（azacitidine）是一种胞苷类似物，可掺入DNA并特异性抑制Dnmts，同时介导蛋白酶体对Dnmts的降解。阿扎胞苷对CpG甲基化及基因转录的下游效应已在诸多研究体系中得到广泛探究，但它对蛋白质组的影响机制仍未被充分阐明。此外，由于其能够特异性诱导Dnmts快速降解（尤其是核心的维持性DNA甲基转移酶Dnmt1），阿扎胞苷可作为一种特异性化学敲低工具，用于研究与Dnmt1相关的功能或物理相互作用组。本研究采用定量蛋白质组学（quantitative proteomics）技术，分析经阿扎胞苷处理的细胞核蛋白质组（nuclear proteome）中蛋白质的降解谱。研究人员鉴定出受阿扎胞苷调控的特定蛋白质、多条信号通路及生物学过程。Dnmt1的相互作用伙伴Uhrf1会发生显著的阿扎胞苷诱导性降解，且该降解过程不依赖于Dnmt1的蛋白表达水平。本研究还鉴定出多种其他染色质相关及表观遗传相关因子，包括含溴结构域的转录调控因子Brd2。实验结果表明，阿扎胞苷可诱导与Dnmt1相关的蛋白质组发生高度特异性的扰动；诸如Uhrf1这类相互作用蛋白对阿扎胞苷敏感，而另一些蛋白（如Dnmt1的相互作用伙伴及稳定性调控因子Usp7）则不受其影响。综上，本研究首次完成了阿扎胞苷敏感型核蛋白质组的全面蛋白质组学分析，并证明5-阿扎胞苷（5-azacitidine）可作为特异性探针，用于探究与Dnmts及染色质相关的蛋白质调控网络。