A high-throughput screen of inactive X chromosome reactivation identifies the enhancement of DNA demethylation by 5-aza-2'-dC upon inhibition of ribonucleotide reductase.

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. The online version of this article (doi:10.1186/s13072-015-0034-4) contains supplementary material, which is available to authorized users.

DNA甲基化对于维持失活X染色体（Xi）上基因的沉默状态非常重要。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 - 氮杂 - 2′ - dC）存在下能重新激活Xi连锁报告基因的小干扰RNA（siRNAs）和化学物质，该抑制剂单独使用时其浓度不足以重新激活Xi。 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 - 氮杂 - 2′ - dC的DNA掺入，从而在全基因组范围内降低DNA甲基化水平，增强了5 - 氮杂 - 2′ - dC的作用。由于5 - 氮杂 - 2′ - dC和RNR抑制剂都用于治疗血液系统恶性肿瘤，我们用5 - 氮杂 - 2′ - dC和RNR抑制剂羟基脲处理髓系白血病细胞系，并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重新激活实验可用于优化药物组合的表观遗传活性。 本文的网络版（doi:10.1186/s13072 - 2015 - 0034 - 4）包含补充材料，授权用户可获取。