Proteomic analysis of azacitidine-induced degradation profiles identifies multiple chromatin and epigenetic regulators including Uhrf1 and Dnmt1 as sensitive to azacitidine

Abstract

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.

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