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Genome-wide mapping of N4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

ABSTRACT: N 4-methylcytosine (4mC) is a natural DNA modification occurring in thermophiles and plays important roles in restriction-modification (R-M) systems in bacterial genomes. However, the precise location and sequence context of 4mC in the whole genome are limited. In this study, we developed an APOBEC3A-mediated deamination sequencing (4mC-AMD-seq) method for genome-wide mapping of 4mC at single-base resolution. In the 4mC-AMD-seq method, cytosine and 5-methylcytosine (5mC) are deaminated by APOBEC3A (A3A) protein to generate uracil and thymine, both of which are read as thymine in sequencing, while 4mC is resistant to deamination and therefore read as cytosine. Thus, the readouts of cytosines from sequencing could manifest the original 4mC sites in genomes. With the 4mC-AMD-seq method, we achieved the genome-wide mapping of 4mC in Deinococcus radiodurans (D. radiodurans). In addition, we confirmed that 4mC, but not 5mC, was the major modification in the D. radiodurans genome. We identified 1586 4mC sites in the genome of D. radiodurans, among which 564 sites were located in the CCGCGG motif. The average methylation levels in the CCGCGG motif and non-CCGCGG sequence were 70.0% and 22.8%, respectively. We envision that the 4mC-AMD-seq method will facilitate the investigation of 4mC functions, including the 4mC-involved R-M systems, in uncharacterized but potentially useful strains.

ORGANISM(S): Deinococcus radiodurans

PROVIDER: GSE189965 | GEO | 2021/12/15

REPOSITORIES: GEO

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Project description:Methylation of cytosines at the 5th carbon position of the aromatic ring has long been regarded as the predominant type of DNA base modification in eukaryotes. Often called “the fifth base”, C5-methylcytosine (5mC) plays an important role in genome defense against mobile genetic elements, and is mostly associated with transcriptional silencing, establishment of the closed chromatin configuration, and repressive histone modifications. Recently, another type of DNA modification, N6-methyladenine (6mA), has been added to the repertoire of modified bases in eukaryotic DNA, and was mostly linked to elevated transcription levels. In prokaryotes, 5mC and 6mA typically constitute components of restriction-modification (R-M) systems, along with N4-methylcytosine (4mC), which so far has been confined to bacteria. Here we report the first case of 4mC occurrence in eukaryotic DNA. We find that bdelloid rotifers, small freshwater invertebrates known for their ability to reproduce clonally and to acquire genes from non-metazoan sources, lack the canonical eukaryotic C5-methyltransferases but instead encode an amino-methyltransferase of bacterial origin, N4CMT, which is present in all bdelloid families separated by tens of millions of years of evolution. The recombinant N4CMT introduces 4mC into genomic DNA in vivo and in vitro. Using SMRT-seq (PRJNA558051), MeDIP-seq, ChIP-seq, and RNA-seq, we examined genome-wide distribution of non-canonical base modifications over annotated genomic features and observed an excess of 4mC in silenced transposable elements and certain tandem repeats, while 6mA tends to associate with transcribed genes and active chromatin. The presence of the chromodomain in N4CMT explains its affinity for repressive histone marks, H3K9me3 and especially H3K27me3. Our results expand the known repertoire of eukaryotic base modifications, shed light on the process of recruitment of methyl groups as epigenetic marks in DNA, and highlight the role of horizontal gene transfer as an important driver of evolutionary innovation in eukaryotes.

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Genome-wide mapping of N4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

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