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

Guan-Zheng Luo,Miao Yu,Ziyang Hao,Kai Chen,Xiaocheng Weng,Chuan He,Fang Wang,Jianzhao Liu,Xin Deng

doi:10.1038/ncomms11301

Guan-Zheng Luo, Miao Yu + Show 7 more

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https://doi.org/10.1038/ncomms11301

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Abstract

Although extensively studied in prokaryotes, the prevalence and significance of DNA N6-methyladenine (6mA or m6dA) 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 N6-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.

Highlights

Extensively studied in prokaryotes, the prevalence and significance of DNA N6-methyladenine (6mA or m6dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark
After treatment with DpnI, long DNA fragments were sonicated to B300 bp in length, and a standard Illumina DNA library was constructed for next-generation sequencing (NGS) sequencing
The G(6mA)TC sites should be cleaved by DpnI, and represented as the reads ends in the sequencing output

Summary

Introduction

Extensively studied in prokaryotes, the prevalence and significance of DNA N6-methyladenine (6mA or m6dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. We reported a restriction enzyme-assisted sequencing approach to identify single 6mA sites in the genome of Chlamydomonas[8]. We show that a 6mA-sensitive restriction enzyme, DpnI, preferentially cleaves duplex DNA at fully methylated sites[24]. A new approach using DpnI obtains the equivalent 6mA map of Chlamydomonas at single-base resolution. DpnI recognizes a canonical GATC sequence motif, and cleaves fully methylated double-stranded DNA at CATC/GATG sites (CATC and GATG are complementary to each other), which further expands the application scope of this method. By combining results using this new approach with previous data, we conclude that most 6mA sites in Chlamydomonas are fully methylated at various sequence a DpnI

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