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Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.

Luo GZ, Wang F, Weng X, Chen K, Hao Z, Yu M, Deng X, Liu J, He C - Nat Commun (2016)

Bottom Line: Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark.DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches.Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.

ABSTRACT
Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N(6)-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

No MeSH data available.


Related in: MedlinePlus

DpnI cleavage assay on fully- or hemi-methylated GATC and CATC/GATG DNA probes.The PAGE gel shows the formation of digested products. (a) DNA probes containing fully methylated GATC (F-GATC, 10 pmol) or hemi-methylated GATC (H-GATC, 10 pmol) were treated with DpnI (10 units) for 30 min or overnight at 37 °C. (b) DNA probes containing fully methylated CATC/GATG (F-CATC, 10 pmol) or hemi-methylated CATC/GATG (H-CATC, 10 pmol) were treated with DpnI for 30 min or overnight. All sequences used are listed in Supplementary Table 1.
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f3: DpnI cleavage assay on fully- or hemi-methylated GATC and CATC/GATG DNA probes.The PAGE gel shows the formation of digested products. (a) DNA probes containing fully methylated GATC (F-GATC, 10 pmol) or hemi-methylated GATC (H-GATC, 10 pmol) were treated with DpnI (10 units) for 30 min or overnight at 37 °C. (b) DNA probes containing fully methylated CATC/GATG (F-CATC, 10 pmol) or hemi-methylated CATC/GATG (H-CATC, 10 pmol) were treated with DpnI for 30 min or overnight. All sequences used are listed in Supplementary Table 1.

Mentions: The previous approach using CviAII- or DpnII-assisted digestion cannot distinguish fully methylated and hemi-methylated sites since these two enzymes are hindered by both fully- or hemi- methylation8. To explore the sensitivity of DpnI to different methylation status, we synthesized single-stranded DNA probes with or without 6mA at specific sequence motifs, and annealed them to afford double-stranded DNA representing fully and hemi-methylated probes. Consistent with the previous report22, DpnI cleaves fully methylated DNA much faster than hemi-methylated DNA containing the GATC motif (Fig. 3a). Specifically, DpnI barely cleaved hemi-methylated CATC/GATG sites, even if the reaction time was excessively prolonged; fully methylated CATC/GATG sites were cleaved by overnight incubation, however (Fig. 3b). We treated the same batch of DNA with DpnI for 30 min or 12 h (overnight), and performed DA-6mA-seq in parallel. Both G(6mA)TC and C(6mA)TC/G(6mA)TG sites in the two data sets concurred (>90% overlapping, Supplementary Fig. 3). These results suggest that the vast majority of the 6mA sites identified by DA-6mA-seq are fully methylated. The additional ∼500 6mA sites that were identified only by using DpnII-based digestion are most likely hemi-methylated8. Genome-wide analysis showed no preferences of these sites to promoters, exons or introns (Supplementary Fig. 3), and the abundances at each sites are low, which may indicate aberrant methylation or roles yet to be identified.


Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.

Luo GZ, Wang F, Weng X, Chen K, Hao Z, Yu M, Deng X, Liu J, He C - Nat Commun (2016)

DpnI cleavage assay on fully- or hemi-methylated GATC and CATC/GATG DNA probes.The PAGE gel shows the formation of digested products. (a) DNA probes containing fully methylated GATC (F-GATC, 10 pmol) or hemi-methylated GATC (H-GATC, 10 pmol) were treated with DpnI (10 units) for 30 min or overnight at 37 °C. (b) DNA probes containing fully methylated CATC/GATG (F-CATC, 10 pmol) or hemi-methylated CATC/GATG (H-CATC, 10 pmol) were treated with DpnI for 30 min or overnight. All sequences used are listed in Supplementary Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835550&req=5

f3: DpnI cleavage assay on fully- or hemi-methylated GATC and CATC/GATG DNA probes.The PAGE gel shows the formation of digested products. (a) DNA probes containing fully methylated GATC (F-GATC, 10 pmol) or hemi-methylated GATC (H-GATC, 10 pmol) were treated with DpnI (10 units) for 30 min or overnight at 37 °C. (b) DNA probes containing fully methylated CATC/GATG (F-CATC, 10 pmol) or hemi-methylated CATC/GATG (H-CATC, 10 pmol) were treated with DpnI for 30 min or overnight. All sequences used are listed in Supplementary Table 1.
Mentions: The previous approach using CviAII- or DpnII-assisted digestion cannot distinguish fully methylated and hemi-methylated sites since these two enzymes are hindered by both fully- or hemi- methylation8. To explore the sensitivity of DpnI to different methylation status, we synthesized single-stranded DNA probes with or without 6mA at specific sequence motifs, and annealed them to afford double-stranded DNA representing fully and hemi-methylated probes. Consistent with the previous report22, DpnI cleaves fully methylated DNA much faster than hemi-methylated DNA containing the GATC motif (Fig. 3a). Specifically, DpnI barely cleaved hemi-methylated CATC/GATG sites, even if the reaction time was excessively prolonged; fully methylated CATC/GATG sites were cleaved by overnight incubation, however (Fig. 3b). We treated the same batch of DNA with DpnI for 30 min or 12 h (overnight), and performed DA-6mA-seq in parallel. Both G(6mA)TC and C(6mA)TC/G(6mA)TG sites in the two data sets concurred (>90% overlapping, Supplementary Fig. 3). These results suggest that the vast majority of the 6mA sites identified by DA-6mA-seq are fully methylated. The additional ∼500 6mA sites that were identified only by using DpnII-based digestion are most likely hemi-methylated8. Genome-wide analysis showed no preferences of these sites to promoters, exons or introns (Supplementary Fig. 3), and the abundances at each sites are low, which may indicate aberrant methylation or roles yet to be identified.

Bottom Line: Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark.DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches.Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.

ABSTRACT
Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N(6)-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

No MeSH data available.


Related in: MedlinePlus