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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

DA-6mA-seq is a highly sensitive method to detect 6mA at specific sites.(a) Quantification of 6mA abundances in Chlamydomonas reinhardtii (Cr), Leishmania major (Lm), Toxoplasma gondii (Tg), Plasmodium falciparum (Pf) and Penicillium chrysogenum (Pc) by LC–MS/MS. Error bars are calculated as the s.d. of three biological replicates. (b) The accumulated nucleotide composition of all the potential 6mA sites identified by DA-6mA-seq in Plasmodium. Sequence logo is generated by WebLogo (ref. 40). (c) The snapshot of genome browser (IGV (ref. 41)) representing a partially methylated site in Plasmodium. The top track shows counts of 5′ ends in the selected region. The ‘Reads' track shows aligned reads. Blue segments represent reads mapped to minus strand and red segments represent reads mapped to plus strand. The red triangle marks GATC site. (d) The snapshot of genome browser representing two completely methylated sites in Chlamydomonas.
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f4: DA-6mA-seq is a highly sensitive method to detect 6mA at specific sites.(a) Quantification of 6mA abundances in Chlamydomonas reinhardtii (Cr), Leishmania major (Lm), Toxoplasma gondii (Tg), Plasmodium falciparum (Pf) and Penicillium chrysogenum (Pc) by LC–MS/MS. Error bars are calculated as the s.d. of three biological replicates. (b) The accumulated nucleotide composition of all the potential 6mA sites identified by DA-6mA-seq in Plasmodium. Sequence logo is generated by WebLogo (ref. 40). (c) The snapshot of genome browser (IGV (ref. 41)) representing a partially methylated site in Plasmodium. The top track shows counts of 5′ ends in the selected region. The ‘Reads' track shows aligned reads. Blue segments represent reads mapped to minus strand and red segments represent reads mapped to plus strand. The red triangle marks GATC site. (d) The snapshot of genome browser representing two completely methylated sites in Chlamydomonas.

Mentions: Previous studies indicate the presence of 6mA in more extensive eukaryotes51027282930, including the human malarial parasite Plasmodium5 and the fungi Penicillium31. To validate the existence of 6mA in these other organisms, we employed liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) to quantify the 6mA abundance in four candidate organisms: Leishmania major, Toxoplasma gondii, Plasmodium falciparum and Penicillium chrysogenum83233 (Fig. 4a); the genomes of Plasmodium and Penicillium were found to contain relatively abundant 6mA (∼0.05–0.15% 6mA/A). We then applied DA-6mA-seq to map 6mA in the genomes of these two organisms. Surprisingly, the distribution patterns of 6mA in the genomes of Plasmodium and Penicillium are very different from that in Chlamydomonas. In Plasmodium, ∼1/5 of GATC sites are extensively methylated all across the genome with methylated CATC or GATG detected at very low level (<1%; Fig. 4b). The extensive G(6mA)TC sites identified are inconsistent with the gross 6mA abundance of the genome. We reasoned that the methylation level of each site might be relatively low; in other words, only a small proportion of individual cells contain 6mA at specific sites, whereas other cells do not. To validate this speculation, we re-analysed the sequencing data and considered the randomly sheared reads overlapping with each GATC sites. If random reads are more than enzyme-digested reads, we defined the site as partially methylated (Fig. 4c). The ratio of random reads to enzyme-digested reads can also help to estimate the percentage of methylation at each specific site8. Under this definition, the majority of G(6mA)TC sites (97%) were methylated with a methylation percentage below 10% in Plasmodium (Supplementary Fig. 4a); in contrast, only 9% of G(6mA)TC sites were partially methylated in the Chlamydomonas genome, which is consistent with much lower 6mA abundance measured in the genome of Plasmodium than that in Chlamydomonas (Fig. 4d and Supplementary Fig. 4a). The wide-spread G(6mA)TC sites are distributed randomly across the genome without any enrichment to gene context (Supplementary Fig. 4b).


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)

DA-6mA-seq is a highly sensitive method to detect 6mA at specific sites.(a) Quantification of 6mA abundances in Chlamydomonas reinhardtii (Cr), Leishmania major (Lm), Toxoplasma gondii (Tg), Plasmodium falciparum (Pf) and Penicillium chrysogenum (Pc) by LC–MS/MS. Error bars are calculated as the s.d. of three biological replicates. (b) The accumulated nucleotide composition of all the potential 6mA sites identified by DA-6mA-seq in Plasmodium. Sequence logo is generated by WebLogo (ref. 40). (c) The snapshot of genome browser (IGV (ref. 41)) representing a partially methylated site in Plasmodium. The top track shows counts of 5′ ends in the selected region. The ‘Reads' track shows aligned reads. Blue segments represent reads mapped to minus strand and red segments represent reads mapped to plus strand. The red triangle marks GATC site. (d) The snapshot of genome browser representing two completely methylated sites in Chlamydomonas.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: DA-6mA-seq is a highly sensitive method to detect 6mA at specific sites.(a) Quantification of 6mA abundances in Chlamydomonas reinhardtii (Cr), Leishmania major (Lm), Toxoplasma gondii (Tg), Plasmodium falciparum (Pf) and Penicillium chrysogenum (Pc) by LC–MS/MS. Error bars are calculated as the s.d. of three biological replicates. (b) The accumulated nucleotide composition of all the potential 6mA sites identified by DA-6mA-seq in Plasmodium. Sequence logo is generated by WebLogo (ref. 40). (c) The snapshot of genome browser (IGV (ref. 41)) representing a partially methylated site in Plasmodium. The top track shows counts of 5′ ends in the selected region. The ‘Reads' track shows aligned reads. Blue segments represent reads mapped to minus strand and red segments represent reads mapped to plus strand. The red triangle marks GATC site. (d) The snapshot of genome browser representing two completely methylated sites in Chlamydomonas.
Mentions: Previous studies indicate the presence of 6mA in more extensive eukaryotes51027282930, including the human malarial parasite Plasmodium5 and the fungi Penicillium31. To validate the existence of 6mA in these other organisms, we employed liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) to quantify the 6mA abundance in four candidate organisms: Leishmania major, Toxoplasma gondii, Plasmodium falciparum and Penicillium chrysogenum83233 (Fig. 4a); the genomes of Plasmodium and Penicillium were found to contain relatively abundant 6mA (∼0.05–0.15% 6mA/A). We then applied DA-6mA-seq to map 6mA in the genomes of these two organisms. Surprisingly, the distribution patterns of 6mA in the genomes of Plasmodium and Penicillium are very different from that in Chlamydomonas. In Plasmodium, ∼1/5 of GATC sites are extensively methylated all across the genome with methylated CATC or GATG detected at very low level (<1%; Fig. 4b). The extensive G(6mA)TC sites identified are inconsistent with the gross 6mA abundance of the genome. We reasoned that the methylation level of each site might be relatively low; in other words, only a small proportion of individual cells contain 6mA at specific sites, whereas other cells do not. To validate this speculation, we re-analysed the sequencing data and considered the randomly sheared reads overlapping with each GATC sites. If random reads are more than enzyme-digested reads, we defined the site as partially methylated (Fig. 4c). The ratio of random reads to enzyme-digested reads can also help to estimate the percentage of methylation at each specific site8. Under this definition, the majority of G(6mA)TC sites (97%) were methylated with a methylation percentage below 10% in Plasmodium (Supplementary Fig. 4a); in contrast, only 9% of G(6mA)TC sites were partially methylated in the Chlamydomonas genome, which is consistent with much lower 6mA abundance measured in the genome of Plasmodium than that in Chlamydomonas (Fig. 4d and Supplementary Fig. 4a). The wide-spread G(6mA)TC sites are distributed randomly across the genome without any enrichment to gene context (Supplementary Fig. 4b).

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