Dataset Information

Uncovering the roles of DNA hemi-methylation in transcriptional regulation using MspJI-assisted hemi-methylation sequencing.

ABSTRACT: Hemi-methylated cytosine dyads widely occur on mammalian genomic DNA, and can be stably inherited across cell divisions, serving as potential epigenetic marks. Previous identification of hemi-methylation relied on harsh bisulfite treatment, leading to extensive DNA degradation and loss of methylation information. Here we introduce Mhemi-seq, a bisulfite-free strategy, to efficiently resolve methylation status of cytosine dyads into unmethylation, strand-specific hemi-methylation, or full-methylation. Mhemi-seq reproduces methylomes from bisulfite-based sequencing (BS-seq & hpBS-seq), including the asymmetric hemi-methylation enrichment flanking CTCF motifs. By avoiding base conversion, Mhemi-seq resolves allele-specific methylation and associated imprinted gene expression more efficiently than BS-seq. Furthermore, we reveal an inhibitory role of hemi-methylation in gene expression and transcription factor (TF)-DNA binding, and some displays a similar extent of inhibition as full-methylation. Finally, we uncover new hemi-methylation patterns within Alu retrotransposon elements. Collectively, Mhemi-seq can accelerate the identification of DNA hemi-methylation and facilitate its integration into the chromatin environment for future studies.

SUBMITTER: Xiong X

PROVIDER: S-EPMC10954476 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Uncovering the roles of DNA hemi-methylation in transcriptional regulation using MspJI-assisted hemi-methylation sequencing.

Xiong Xiong X Chen Hengye H Zhang Qifan Q Liu Yangying Y Xu Chenhuan C

Nucleic acids research 20240301 5

Hemi-methylated cytosine dyads widely occur on mammalian genomic DNA, and can be stably inherited across cell divisions, serving as potential epigenetic marks. Previous identification of hemi-methylation relied on harsh bisulfite treatment, leading to extensive DNA degradation and loss of methylation information. Here we introduce Mhemi-seq, a bisulfite-free strategy, to efficiently resolve methylation status of cytosine dyads into unmethylation, strand-specific hemi-methylation, or full-methyla ...[more]

PMID: 38261991

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Project description:BackgroundFragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine.ResultsA semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2'-deoxycytidine-5'-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2'-deoxycytidine-5'-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation.ConclusionsThe proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing.

Dataset Information

Uncovering the roles of DNA hemi-methylation in transcriptional regulation using MspJI-assisted hemi-methylation sequencing.

Publications

Uncovering the roles of DNA hemi-methylation in transcriptional regulation using MspJI-assisted hemi-methylation sequencing.

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