Abstract
Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) inthe regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically thehighly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modificationas a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.
Highlights
Identification of N6-mA DNA Modifications in Human Glioblastoma To investigate the roles of the N6-mA DNA modification in human glioblastoma, we extracted genomic DNA from functionally validated human patient-derived glioblastoma stem cell (GSC) models (387, D456, GSC23, and 1919) and primary human tumor specimens (3028 and CW2386) and performed dot blot analysis using an N6-mA-specific antibody that has been validated previously (Greer et al, 2015; Wu et al, 2016; Zhang et al, 2015)
N6-mA levels were independently quantified by mass spectrometry (MS) using an established and highly sensitive MS approach with stable isotope-labeled [15N5] N6-mA as an internal standard for sample enrichment and quantification (Figures S1A and S1B), which demonstrated that N6-mA levels in GSCs were elevated more than 100-fold compared with normal human astrocytes (Figure 1B)
Effects of cell culture conditions were ruled out using DNA N6-mA immunohistochemistry on a tissue microarray (TMA) with normal brain and glioblastoma tissues, confirming increased DNA N6-mA levels in glioblastoma (Figures 1E and 1F)
Summary
Enzymatic hydrolysis of DNA The enzymatic digestion of DNA was performed as described here. One mg of genomic DNA was first spiked with uniformly 15N-labeled 20-deoxyadenosine and D3-labeled N6-methyl-20-deoxyadenosine. DNA hydrolysis was performed with the addition of nuclease P1 (0.1 U), phosphodiesterase 2 (0.000125 U), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (0.25 nmol) and 4.5 mL solution containing 300 mM sodium acetate (pH 5.6) and 10 mM zinc chloride to the DNA solution. Incubation was carried out at 37C for 24 hours. A second step digestion was carried out with 0.1 unit of alkaline phosphatase, 0.00025 unit phosphodiesterase I, 6 mL of 0.5 M Tris-HCl buffer (pH 8.9) at 37C for 2 hours. The digestion mixture was neutralized by the addition of formic acid and the enzymes in the digestion mixture were removed by chloroform extraction
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