Minkovsky Alissa, Sahakyan Anna, Bonora Giancarlo, Damoiseaux Robert, Dimitrova Elizabeth, Rubbi Liudmilla, Pellegrini Matteo, Radu Caius G, Plath Kathrin
Department of Biological Chemistry, David Geffen School of Medicine, Eli and Edythe Broad Center for Regenerative Medicine, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, 615 Charles E. Young Drive South, BSRB 390D, Los Angeles, 90095 USA.
Department of Molecular and Medicinal Chemistry and California NanoSystems Institute, University of California, Los Angeles, 90095 USA.
Epigenetics Chromatin. 2015 Oct 13;8:42. doi: 10.1186/s13072-015-0034-4. eCollection 2015.
DNA methylation is important for the maintenance of the silent state of genes on the inactive X chromosome (Xi). Here, we screened for siRNAs and chemicals that reactivate an Xi-linked reporter in the presence of 5-aza-2'-deoxycytidine (5-aza-2'-dC), an inhibitor of DNA methyltransferase 1, at a concentration that, on its own, is not sufficient for Xi-reactivation.
We found that inhibition of ribonucleotide reductase (RNR) induced expression of the reporter. RNR inhibition potentiated the effect of 5-aza-2'-dC by enhancing its DNA incorporation, thereby decreasing DNA methylation levels genome-wide. Since both 5-aza-2'-dC and RNR-inhibitors are used in the treatment of hematological malignancies, we treated myeloid leukemia cell lines with 5-aza-2'-dC and the RNR-inhibitor hydroxyurea, and observed synergistic inhibition of cell growth and a decrease in genome-wide DNA methylation.
Taken together, our study identifies a drug combination that enhances DNA demethylation by altering nucleotide metabolism. This demonstrates that Xi-reactivation assays can be used to optimize the epigenetic activity of drug combinations.
DNA甲基化对于维持失活X染色体（Xi）上基因的沉默状态至关重要。在此，我们筛选了在DNA甲基转移酶1抑制剂5-氮杂-2'-脱氧胞苷（5-aza-2'-dC）存在下重新激活Xi连锁报告基因的小干扰RNA（siRNA）和化学物质，5-aza-2'-dC单独使用时不足以重新激活Xi，但其浓度可用于本实验。
我们发现抑制核糖核苷酸还原酶（RNR）可诱导报告基因的表达。RNR抑制通过增强5-aza-2'-dC的DNA掺入来增强其作用，从而降低全基因组的DNA甲基化水平。由于5-aza-2'-dC和RNR抑制剂均用于治疗血液系统恶性肿瘤，我们用5-aza-2'-dC和RNR抑制剂羟基脲处理髓系白血病细胞系，观察到细胞生长的协同抑制以及全基因组DNA甲基化的降低。
综上所述，我们的研究确定了一种通过改变核苷酸代谢来增强DNA去甲基化的药物组合。这表明Xi重新激活试验可用于优化药物组合的表观遗传活性。
