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Epigenetics Chromatin. 2015 Oct 13;8:42. doi: 10.1186/s13072-015-0034-4. eCollection 2015.

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.

Author information

1
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.
2
Department of Molecular and Medicinal Chemistry and California NanoSystems Institute, University of California, Los Angeles, 90095 USA.
3
Ahmanson Translational Imaging Division, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, 90095 USA.
4
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 90095 USA.
#
Contributed equally

Abstract

BACKGROUND:

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.

RESULTS:

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.

CONCLUSIONS:

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.

KEYWORDS:

5-aza-2′-dC; DNA methylation; Hydroxyurea; Ribonucleotide reductase; X chromosome inactivation

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