Existence and possible roles of independent non-CpG methylation in the mammalian brain

Methylated non-CpGs (mCpHs) in mammalian cells yield weak enrichment signals and colocalize with methylated CpGs (mCpGs), thus have been considered byproducts of hyperactive methyltransferases. However, mCpHs are cell type-specific and associated with epigenetic regulation, although their dependency on mCpGs remains to be elucidated. In this study, we demonstrated that mCpHs colocalize with mCpGs in pluripotent stem cells, but not in brain cells. In addition, profiling genome-wide methylation patterns using a hidden Markov model revealed abundant genomic regions in which CpGs and CpHs are differentially methylated in brain. These regions were frequently located in putative enhancers, and mCpHs within the enhancers increased in correlation with brain age. The enhancers with hypermethylated CpHs were associated with genes functionally enriched in immune responses, and some of the genes were related to neuroinflammation and degeneration. This study provides insight into the roles of non-CpG methylation as an epigenetic code in the mammalian brain genome.     

Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection

Whole genome base-resolution methylome sequencing allows for the most comprehensive analysis of DNA methylation, however, the considerable sequencing cost often limits its applications. While reduced representation sequencing can be an affordable alternative, over 80% of CpGs in the genome are not covered. Building on our recently developed TET-assisted pyridine borane sequencing (TAPS) method, we here described endonuclease enrichment TAPS (eeTAPS), which utilizes dihydrouracil (DHU)-cleaving endonuclease digestion of TAPS-converted DNA to enrich methylated CpG sites (mCpGs). eeTAPS can accurately detect 87% of mCpGs in the mouse genome with a sequencing depth equivalent to 4× whole genome sequencing. In comparison, reduced representation TAPS (rrTAPS) detected less than 4% of mCpGs with 2.5× sequencing depth. Our results demonstrate eeTAPS to be a new strategy for cost-effective genome-wide methylation analysis at single-CpG resolution that can fill the gap between whole-genome and reduced representation sequencing.     

CpG_MPs: identification of CpG methylation patterns of genomic regions from high-throughput bisulfite sequencing data

High-throughput bisulfite sequencing is widely used to measure cytosine methylation at single-base resolution in eukaryotes. It permits systems-level analysis of genomic methylation patterns associated with gene expression and chromatin structure. However, methods for large-scale identification of methylation patterns from bisulfite sequencing are lacking. We developed a comprehensive tool, CpG_MPs, for identification and analysis of the methylation patterns of genomic regions from bisulfite sequencing data. CpG_MPs first normalizes bisulfite sequencing reads into methylation level of CpGs. Then it identifies unmethylated and methylated regions using the methylation status of neighboring CpGs by hotspot extension algorithm without knowledge of pre-defined regions. Furthermore, the conservatively and differentially methylated regions across paired or multiple samples (cells or tissues) are identified by combining a combinatorial algorithm with Shannon entropy. CpG_MPs identified large amounts of genomic regions with different methylation patterns across five human bisulfite sequencing data during cellular differentiation. Different sequence features and significantly cell-specific methylation patterns were observed. These potentially functional regions form candidate regions for functional analysis of DNA methylation during cellular differentiation. CpG_MPs is the first user-friendly tool for identifying methylation patterns of genomic regions from bisulfite sequencing data, permitting further investigation of the biological functions of genome-scale methylation patterns.     

A nuclear protein that binds preferentially to methylated DNA in vitro may play a role in the inaccessibility of methylated CpGs in mammalian nuclei

The effects of DNA methylation on gene expression and chromatin structure suggest the existence of a mechanism in the nucleus capable of distinguishing methylated and non-methylated sequences. We report the finding of a nuclear protein in several vertebrate tissues and cell lines that binds preferentially to methylated DNA in vitro. Its lack of sequence-specific requirements makes it potentially capable of binding to any methylated sequence in mammalian nuclei. An in vivo counterpart of these results is that methylated CpGs are inaccessible to nucleases within nuclei. In contrast, non-methylated CpG sites, located mainly at CpG islands, and restriction sites not containing this dinucleotide, are relatively accessible. The possibility that DNA methylation acts through binding to specific proteins that could alter chromatin structure is discussed.     

Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.

In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.