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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

Non-GATC 6mA sites in the Chlamydomonas genome identified by DA-6mA-seq.(a) The accumulated nucleotide composition of all potential 6mA sites identified by DA-6mA-seq and computation. Sequence logo is generated by WebLogo (ref. 40). The height represents the significance of enrichment of each nucleotide at that position. (b) Percentages of 6mA sites at different tetramer sequence contexts. (c) DpnI effectively cleaves the fully methylated GATC (F-GATC) and CATC (F-CATC) sites but does not digest unmethylated sites (GATC, CATC). Probes (5 pmol) were treated with DpnI (10 units) overnight at 37 °C. (d) The periodic distribution pattern of methylated CATC/GATG sites around transcription start sites (TSS) is nearly identical to the pattern of methylated GATC sites.
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f2: Non-GATC 6mA sites in the Chlamydomonas genome identified by DA-6mA-seq.(a) The accumulated nucleotide composition of all potential 6mA sites identified by DA-6mA-seq and computation. Sequence logo is generated by WebLogo (ref. 40). The height represents the significance of enrichment of each nucleotide at that position. (b) Percentages of 6mA sites at different tetramer sequence contexts. (c) DpnI effectively cleaves the fully methylated GATC (F-GATC) and CATC (F-CATC) sites but does not digest unmethylated sites (GATC, CATC). Probes (5 pmol) were treated with DpnI (10 units) overnight at 37 °C. (d) The periodic distribution pattern of methylated CATC/GATG sites around transcription start sites (TSS) is nearly identical to the pattern of methylated GATC sites.

Mentions: DpnI efficiently cleaves G(6mA)TC sites based on previous reportes2224. However, our sequencing results have revealed cleavage of a large fraction of methylated non-GATC sites (Fig. 2a). Specifically, 76% of the identified 6mA sites are located in the CATC/GATG context while only 19% are located in the GATC context (Fig. 2b). This high proportion of non-GATC 6mA sites prompted us to confirm the cleavage of C(6mA)TC by DpnI in addition to G(6mA)TC. In fact, a previous report suggested that DpnI could also recognize motifs other than GATC (ref. 25). We synthesized oligonucleotides with 6mA site in the CATC/GATG context, and annealed them to form double-stranded DNA. After treatment with DpnI, the DNA probes with C(6mA)TC and G(6mA)TG motifs were explicitly cleaved at the 6mA site after 12 h of incubation (Fig. 2c). As negative controls, double-stranded DNA probes containing unmethylated GATC or CATC were resistant to cleavage even with overnight incubation. Therefore, the non-GATC sites identified by DA-6mA-seq should be genuine 6mA sites. Indeed, these sites overlap very well with the previous 6mA map determined by using IP followed by sequencing (Supplementary Fig. 2a)8. We then inspected the genomic distribution of these non-GATC sites, and found that they were highly enriched at promoter regions (Supplementary Fig. 2b). Furthermore, these 6mA sites form a clear periodic pattern around transcription start sites, which is almost identical to the pattern of G(6mA)TC in the same genomic DNA (Fig. 2d).


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)

Non-GATC 6mA sites in the Chlamydomonas genome identified by DA-6mA-seq.(a) The accumulated nucleotide composition of all potential 6mA sites identified by DA-6mA-seq and computation. Sequence logo is generated by WebLogo (ref. 40). The height represents the significance of enrichment of each nucleotide at that position. (b) Percentages of 6mA sites at different tetramer sequence contexts. (c) DpnI effectively cleaves the fully methylated GATC (F-GATC) and CATC (F-CATC) sites but does not digest unmethylated sites (GATC, CATC). Probes (5 pmol) were treated with DpnI (10 units) overnight at 37 °C. (d) The periodic distribution pattern of methylated CATC/GATG sites around transcription start sites (TSS) is nearly identical to the pattern of methylated GATC sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Non-GATC 6mA sites in the Chlamydomonas genome identified by DA-6mA-seq.(a) The accumulated nucleotide composition of all potential 6mA sites identified by DA-6mA-seq and computation. Sequence logo is generated by WebLogo (ref. 40). The height represents the significance of enrichment of each nucleotide at that position. (b) Percentages of 6mA sites at different tetramer sequence contexts. (c) DpnI effectively cleaves the fully methylated GATC (F-GATC) and CATC (F-CATC) sites but does not digest unmethylated sites (GATC, CATC). Probes (5 pmol) were treated with DpnI (10 units) overnight at 37 °C. (d) The periodic distribution pattern of methylated CATC/GATG sites around transcription start sites (TSS) is nearly identical to the pattern of methylated GATC sites.
Mentions: DpnI efficiently cleaves G(6mA)TC sites based on previous reportes2224. However, our sequencing results have revealed cleavage of a large fraction of methylated non-GATC sites (Fig. 2a). Specifically, 76% of the identified 6mA sites are located in the CATC/GATG context while only 19% are located in the GATC context (Fig. 2b). This high proportion of non-GATC 6mA sites prompted us to confirm the cleavage of C(6mA)TC by DpnI in addition to G(6mA)TC. In fact, a previous report suggested that DpnI could also recognize motifs other than GATC (ref. 25). We synthesized oligonucleotides with 6mA site in the CATC/GATG context, and annealed them to form double-stranded DNA. After treatment with DpnI, the DNA probes with C(6mA)TC and G(6mA)TG motifs were explicitly cleaved at the 6mA site after 12 h of incubation (Fig. 2c). As negative controls, double-stranded DNA probes containing unmethylated GATC or CATC were resistant to cleavage even with overnight incubation. Therefore, the non-GATC sites identified by DA-6mA-seq should be genuine 6mA sites. Indeed, these sites overlap very well with the previous 6mA map determined by using IP followed by sequencing (Supplementary Fig. 2a)8. We then inspected the genomic distribution of these non-GATC sites, and found that they were highly enriched at promoter regions (Supplementary Fig. 2b). Furthermore, these 6mA sites form a clear periodic pattern around transcription start sites, which is almost identical to the pattern of G(6mA)TC in the same genomic DNA (Fig. 2d).

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