CpG Context Research Articles

Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression

Like the nucleus, mitochondria contain their own DNA and recent reports provide accumulating evidence that also the mitochondrial DNA (mtDNA) is subjective to DNA methylation. This evidence includes the demonstration of mitochondria-localised DNA methyltransferases and demethylases, and the detection of mtDNA methylation as well as hydroxymethylation. Importantly, differential mtDNA methylation has been linked to aging and diseases, including cancer and diabetes. However, functionality of mtDNA methylation has not been demonstrated. Therefore, we targeted DNA methylating enzymes (modifying cytosine in the CpG or GpC context) to the mtDNA. Unexpectedly, mtDNA gene expression remained unchanged upon induction of CpG mtDNA methylation, whereas induction of C-methylation in the GpC context decreased mtDNA gene expression. Intriguingly, in the latter case, the three mtDNA promoters were differentially affected in each cell line, while cellular function seemed undisturbed. In conclusion, this is the first study which directly addresses the potential functionality of mtDNA methylation. Giving the important role of mitochondria in health and disease, unravelling the impact of mtDNA methylation adds to our understanding of the role of mitochondria in physiological and pathophysiological processes.

A/T Run Geometry of B-form DNA Is Independent of Bound Methyl-CpG Binding Domain, Cytosine Methylation and Flanking Sequence

DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed.

TET2-mediated 5-hydroxymethylcytosine induces genetic instability and mutagenesis.

The family of Ten-Eleven Translocation (TET) proteins is implicated in the process of active DNA demethylation and thus in epigenetic regulation. TET 1, 2 and 3 proteins are oxygenases that can hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). The base excision repair (BER) pathway removes the resulting 5-fC and 5-caC bases paired with a guanine and replaces them with regular cytosine. The question arises whether active modification of 5-mC residues and their subsequent elimination could affect the genomic DNA stability. Here, we generated two inducible cell lines (Ba/F3-EPOR, and UT7) overexpressing wild-type or catalytically inactive human TET2 proteins. Wild-type TET2 induction resulted in an increased level of 5-hmC and a cell cycle defect in S phase associated with higher level of phosphorylated P53, chromosomal and centrosomal abnormalities. Furthermore, in a thymine-DNA glycosylase (Tdg) deficient context, the TET2-mediated increase of 5-hmC induces mutagenesis characterized by GC>AT transitions in CpG context suggesting a mutagenic potential of 5-hmC metabolites. Altogether, these data suggest that TET2 activity and the levels of 5-hmC and its derivatives should be tightly controlled to avoid genetic and chromosomal instabilities. Moreover, TET2-mediated active demethylation might be a very dangerous process if used to entirely demethylate the genome and might rather be used only at specific loci.

5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA.

CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types.

Preservation of methylated CpG dinucleotides in human CpG islands.

BackgroundCpG dinucleotides are extensively underrepresented in mammalian genomes. It is widely accepted that genome-wide CpG depletion is predominantly caused by an elevated CpG > TpG mutation rate due to frequent cytosine methylation in the CpG context. Meanwhile the CpG content in genomic regions called CpG islands (CGIs) is noticeably higher. This observation is usually explained by lower CpG > TpG substitution rates within CGIs due to reduced cytosine methylation levels.ResultsBy combining genome-wide data on substitutions and methylation levels in several human cell types we have shown that cytosine methylation in human sperm cells was strongly and consistently associated with increased CpG > TpG substitution rates. In contrast, this correlation was not observed for embryonic stem cells or fibroblasts. Surprisingly, the decreased sperm CpG methylation level was insufficient to explain the reduced CpG > TpG substitution rates in CGIs.ConclusionsWhile cytosine methylation in human sperm cells is strongly associated with increased CpG > TpG substitution rates, substitution rates are significantly reduced within CGIs even after sperm CpG methylation levels and local GC content are controlled for. Our findings are consistent with strong negative selection preserving methylated CpGs within CGIs including intergenic ones.ReviewersReviewed by: Vladimir Kuznetsov, Shamil Sunyaev, Alexey KondrashovElectronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0113-x) contains supplementary material, which is available to authorized users.

Focussing reduced representation CpG sequencing through judicious restriction enzyme choice

Current restriction enzyme based reduced representation methylation analyses aim for limited, but unbiased, methylome coverage. As the current best estimate suggests that only ~20% of CpGs are dynamically regulated, we characterised the CpG and genomic context surrounding all suitable restriction enzyme sites to identify those that were located in regions rich in dynamically methylated CpGs. The restriction-site distributions for MspI, BstUI, and HhaI were non-random. CpGs in CGI and shelf+shore could be enriched, particularly in gene bodies for all genomic regions, promoters (TSS1500, TSS200), intra- (1st exon, gene body, 3′UTR, 5′UTR) and inter-genic regions. HpyCH4IV enriched CpG elements in the open sea for all genomic elements. Judicious restriction enzyme choice improves the focus of reduced representation approaches by avoiding the monopolization of read coverage by genomic regions that are irrelevant, unwanted or difficult to map, and only sequencing the most informative fraction of CpGs.

An evaluation of methods to test predefined genomic regions for differential methylation in bisulfite sequencing data.

In the biology of tissue development and diseases, DNA methylation plays an important role. For a deeper understanding, it is crucial to accurately compare DNA methylation patterns between groups of samples representing different conditions. A widely used method to investigate DNA methylation in the CpG context is bisulfite sequencing, which produces data on the single-nucleotide scale. While there are benefits to analyzing CpG sites on a basepair level, there are both biological and statistical reasons to test entire genomic regions for differential methylation. However, the analysis of DNA methylation is hampered by the lack of best practice standards. Here, we compared multiple approaches for testing predefined genomic regions for differential DNA methylation in bisulfite sequencing data. Nine methods were evaluated: BiSeq, COHCAP, Goeman's Global Test, Limma, methylKit/eDMR, RADMeth and three log-linear regression approaches with different distribution assumptions. We applied these methods to simulated data and determined their sensitivity and specificity. This revealed performance differences, which were also seen when applied to real data. Methods that first test single CpG sites and then test regions based on transformed CpG-wise P-values performed better than methods that summarize methylation levels or raw reads. Interestingly, smoothing of methylation levels had a negligible impact. In particular, Global Test, BiSeq and RADMeth/z-test outperformed the other methods we evaluated, providing valuable guidance for more accurate analysis of DNA methylation.

MethylPipe and compEpiTools: a suite of R packages for the integrative analysis of epigenomics data.

BackgroundNumerous methods are available to profile several epigenetic marks, providing data with different genome coverage and resolution. Large epigenomic datasets are then generated, and often combined with other high-throughput data, including RNA-seq, ChIP-seq for transcription factors (TFs) binding and DNase-seq experiments. Despite the numerous computational tools covering specific steps in the analysis of large-scale epigenomics data, comprehensive software solutions for their integrative analysis are still missing. Multiple tools must be identified and combined to jointly analyze histone marks, TFs binding and other -omics data together with DNA methylation data, complicating the analysis of these data and their integration with publicly available datasets.ResultsTo overcome the burden of integrating various data types with multiple tools, we developed two companion R/Bioconductor packages. The former, methylPipe, is tailored to the analysis of high- or low-resolution DNA methylomes in several species, accommodating (hydroxy-)methyl-cytosines in both CpG and non-CpG sequence context. The analysis of multiple whole-genome bisulfite sequencing experiments is supported, while maintaining the ability of integrating targeted genomic data. The latter, compEpiTools, seamlessly incorporates the results obtained with methylPipe and supports their integration with other epigenomics data. It provides a number of methods to score these data in regions of interest, leading to the identification of enhancers, lncRNAs, and RNAPII stalling/elongation dynamics. Moreover, it allows a fast and comprehensive annotation of the resulting genomic regions, and the association of the corresponding genes with non-redundant GeneOntology terms. Finally, the package includes a flexible method based on heatmaps for the integration of various data types, combining annotation tracks with continuous or categorical data tracks.ConclusionsmethylPipe and compEpiTools provide a comprehensive Bioconductor-compliant solution for the integrative analysis of heterogeneous epigenomics data. These packages are instrumental in providing biologists with minimal R skills a complete toolkit facilitating the analysis of their own data, or in accelerating the analyses performed by more experienced bioinformaticians.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0742-6) contains supplementary material, which is available to authorized users.

A Sensitive Approach to Map Genome-wide 5-Hydroxymethylcytosine and 5-Formylcytosine at Single-Base Resolution

Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biologicalfunctions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC chemical labeling enrichment method. The sensitive method enables detection of low-abundance 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated agenome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC in non-CpG context exhibit lower abundance, more dynamically, than those in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H= A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite conversion-free method can be applied to biological samples with limited starting material or low-abundance cytosine modifications.

Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation.

BackgroundStudies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc.ResultsHomologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster’s mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5’-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms.ConclusionsComparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these 'relatively young’ genes was critical for the origin and radiation of eukaryotes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1119) contains supplementary material, which is available to authorized users.

Pyrene‐Based Quantitative Detection of the 5‐Formylcytosine Loci Symmetry in the CpG Duplex Content during TET‐Dependent Demethylation

AbstractMethylcytosine (5mC) is mostly symmetrically distributed in CpG sites. Ten‐eleven‐translocation (TET) proteins are the key enzymes involved in active DNA demethylation through stepwise oxidation of 5mC. However, oxidation pathways of TET enzymes in the symmetrically methylated CpG context are still elusive. Employing the unique fluorescence properties of pyrene group, we designed and synthesized a sensitive fluorescence‐based probe not only to target 5‐formylcytosine (5fC) sites, but also to distinguish symmetric from asymmetric 5fC sites in the double stranded DNA context during TET‐dependent 5mC oxidation process. Using this novel probe, we revealed dominant levels of symmetric 5fC among total 5fC sites during in vitro TET‐dependent 5mC oxidation and novel mechanistic insights into the TET‐dependent 5mC oxidation in the mCpG context.

Pyrene-based quantitative detection of the 5-formylcytosine loci symmetry in the CpG duplex content during TET-dependent demethylation.

Methylcytosine (5mC) is mostly symmetrically distributed in CpG sites. Ten-eleven-translocation (TET) proteins are the key enzymes involved in active DNA demethylation through stepwise oxidation of 5mC. However, oxidation pathways of TET enzymes in the symmetrically methylated CpG context are still elusive. Employing the unique fluorescence properties of pyrene group, we designed and synthesized a sensitive fluorescence-based probe not only to target 5-formylcytosine (5fC) sites, but also to distinguish symmetric from asymmetric 5fC sites in the double stranded DNA context during TET-dependent 5mC oxidation process. Using this novel probe, we revealed dominant levels of symmetric 5fC among total 5fC sites during in vitro TET-dependent 5mC oxidation and novel mechanistic insights into the TET-dependent 5mC oxidation in the mCpG context.

Aberrant repair initiated by mismatch-specific thymine-DNA glycosylases provides a mechanism for the mutational bias observed in CpG islands.

The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N6-ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes.

Crystal Structure of TET2-DNA Complex: Insight into TET-Mediated 5mC Oxidation

TET proteins oxidize 5-methylcytosine (5mC) on DNA and play important roles in various biological processes. Mutations of TET2 are frequently observed in myeloid malignance. Here, we present the crystal structure of human TET2 bound to methylated DNA at 2.02Å resolution. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. 5mC is inserted into the catalytic cavity with the methyl group orientated to catalytic Fe(II) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(II)/NOG-chelating, DNA-interacting, and zinc-chelating residues are frequently observed in human cancers. Our studies provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation.

DNA modifications repaired by base excision repair are epigenetic

CREB controls ∼25% of the mammalian transcriptome. Small changes in binding to its consensus (CRE) sequence are likely to be amplified many fold in initiating transcription. Here we show that DNA lesions repaired by the base excision repair (BER) pathway modulate CREB binding to CRE. We generated Kd values by electrophoretic mobility shift assays using purified human CREB and a 39-mer double-stranded oligonucleotide containing modified or wild-type CRE. CRE contains two guanine residues per strand, one in a CpG islet. Alterations in CRE resulted in positive or negative changes in Kd over two orders of magnitude depending on location and modification. Cytosine methylation or oxidation of both guanines greatly diminished binding; a G/U mispair in the CpG context enhanced binding. Intermediates in the BER pathway at one G residue or the other resulted in reduced binding, depending on the specific location, while there was no change in binding when the single G residue outside of the CpG islet was oxidized. CREB recruits other partners after dimers form on DNA. Only UpG increased DNA.CREB dimer formation. Since oxidation is ongoing and conversion of cytosine to uracil occurs spontaneously or at specific times during differentiation and development, we propose that BER substrates are epigenetic and modulate transcription factor recognition/binding.

GENOME-WIDE ANALYSIS AND MODELING OF DNA METHYLATION SUSCEPTIBILITY IN 30 BREAST CANCER CELL LINES BY USING CpG FLANKING SEQUENCES

DNA methylation is an epigenetic modification of DNA that adds a methyl group to cytosine. Aberrant DNA methylation in the CpG context is frequently observed in cancer cells and it is known that aberrant DNA methylation silences tumor repressor genes. However, the mechanism of DNA methylation is not well understood. A widely accepted hypothesis is that DNA methylation does not randomly occur and may be controlled by some instructive mechanisms. In this paper, we conducted an extensive study on this important question by using proprietary sequencing data from methyl-binding domain protein (MBD)-Cap ChIP sequencing experiments for 30 breast cancer cell lines. The goal of our study is to investigate difference in nucleotide composition around CpG sites, where high levels of methylation are observed, and use the information for modeling DNA methylation susceptibility. First, we observed that DNA methylation is not uniform in the whole-genome region and also that the character composition of CpG flanking sequences are significantly different between hyper- and hypo-methylated groups. In an in-depth study, we used information theoretic approaches such as entropy and relative entropy to delineate character composition features and found enrichment of A (Adenine) and T (Thymine) in specific positions around hyper-methylated sites. As the methylation level is increased, A, T proportions in specific positions around hypermethylated sites are increased while A, T proportions in other positions around hypermethylated sites are decreased. Second, we built predictive models for methylation susceptibility by using characters flanking CpG sites as features and hyper-/hypo-methylation status as class. Third, we constructed predictive models using a log odds score of two profiles from DNA sequences surrounding CpG sites of hyper- and hypo-methylated groups. This analysis showed that distribution of profile scores of hyper-/hypo-methylated sites sequences is quite distinct. Our genome-wide CpG methylation study shows that nucleotides around CpG sites caries information for cytosine methylation. This is consistent with the seminal work on the instructive evidence of DNA methylation by Keshet et al. (Nature Genetics, 38(2), 149-153 2006). Our study is on the full genome scale and used the sequencing data, thus our study is significantly different in terms of resolution of data and analysis methods used for the study by Keshet et al.

DNA Methylation and Cancer Development: Molecular Mechanism

DNA methylation is a significant regulator of gene expression, and its role in carcinogenesis recently has been a subject of remarkable interest. The aim of this review is to analyze the mechanism and cell regulatory effects of both hypo- and hyper-DNA methylation on cancer. In this review, we report new developments and their implications regarding the effects of DNA methylation on cancer development. Indeed, alteration of the pattern of DNA methylation has been a constant finding in cancer cells of the same type and differences in the pattern of DNA methylation not only occur in a variety of tumor types, but also in developmental processes Furthermore, the pattern of histone modification appears to be a predicator of the risk of recurrence of human cancers. It is well known that hypermethylation represses transcription of the promoter sections of tumor-suppressor genes leading to gene silencing. However, hypomethylation also has been identified as a cause of oncogenesis. Furthermore, experiments concerning the mechanism of methylation and its control have led to the discovery of many regulatory enzymes and proteins. This review reports on methods developed for the detection of 5-hydroxymethylcytosine methylation at the 5-methylcytosine of protein domains in the CpG context compared to non-methylated DNA, histone modification, and microRNA change.

The Mammalian de Novo DNA Methyltransferases DNMT3A and DNMT3B Are Also DNA 5-Hydroxymethylcytosine Dehydroxymethylases

For cytosine (C) demethylation of vertebrate DNA, it is known that the TET proteins could convert 5-methyl C (5-mC) to 5-hydroxymethyl C (5-hmC). However, DNA dehydroxymethylase(s), or enzymes able to directly convert 5-hmC to C, have been elusive. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are also redox-dependent DNA dehydroxymethylases. Significantly, intactness of the C methylation catalytic sites of these de novo enzymes is also required for their 5-hmC dehydroxymethylation activity. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation.

Crystal Structure of Human Methyl-Binding Domain IV Glycosylase Bound to Abasic DNA

The mammalian repair protein MBD4 (methyl-CpG-binding domain IV) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC), and downstream base excision repair proteins restore a G·C pair. MBD4 is also implicated in active DNA demethylation by initiating base excision repair of G·T mispairs generated by a deaminase enzyme. The question of how mismatch glycosylases attain specificity for excising thymine from G·T, but not A·T, pairs remains largely unresolved. Here, we report a crystal structure of the glycosylase domain of human MBD4 (residues 427–580) bound to DNA containing an abasic nucleotide paired with guanine, providing a glimpse of the enzyme–product complex. The mismatched guanine remains intrahelical, nestled into a recognition pocket. MBD4 provides selective interactions with the mismatched guanine (N1H, N2H2) that are not compatible with adenine, which likely confer mismatch specificity. The structure reveals no interactions that would be expected to provide the MBD4 glycosylase domain with specificity for acting at CpG sites. Accordingly, we find modest 1.5- to 2.7-fold reductions in G·T activity upon altering the CpG context. In contrast, 37- to 580-fold effects were observed previously for thymine DNA glycosylase. These findings suggest that specificity of MBD4 for acting at CpG sites depends largely on its methyl-CpG-binding domain, which binds preferably to G·T mispairs in a methylated CpG site. MBD4 glycosylase cannot excise 5-formylcytosine (fC) or 5-carboxylcytosine (caC), intermediates in a Tet (ten eleven translocation)-initiated DNA demethylation pathway. Our structure suggests that MBD4 does not provide the electrostatic interactions needed to excise these oxidized forms of mC.

Role of CpG context and content in evolutionary signatures of brain DNA methylation

DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.