Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

https://www.pnas.org/content/pnas/93/18/9821.full.pdf

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands

Here we report that increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers altered the offspring epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus. Offspring of mothers that showed high levels of LG and ABN were found to have differences in DNA methylation, as compared to offspring of 'low-LG-ABN' mothers. These differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Central infusion of a histone deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding, GR expression and hypothalamic-pituitary-adrenal (HPA) responses to stress, suggesting a causal relation among epigenomic state, GR expression and the maternal effect on stress responses in the offspring. Thus we show that an epigenomic state of a gene can be established through behavioral programming, and it is potentially reversible.

/pdf/epigenetic-programming-by-maternal-behavior-nhsklwyk7o.pdf

Epigenetic programming by maternal behavior.

Maternal care influences hypothalamic-pituitary-adrenal (HPA) function in the rat through epigenetic programming of glucocorticoid receptor expression. In humans, childhood abuse alters HPA stress responses and increases the risk of suicide. We examined epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls. We found decreased levels of glucocorticoid receptor mRNA, as well as mRNA transcripts bearing the glucocorticoid receptor 1F splice variant and increased cytosine methylation of an NR3C1 promoter. Patch-methylated NR3C1 promoter constructs that mimicked the methylation state in samples from abused suicide victims showed decreased NGFI-A transcription factor binding and NGFI-A–inducible gene transcription. These findings translate previous results from rat to humans and suggest a common effect of parental care on the epigenetic regulation of hippocampal glucocorticoid receptor expression.

/pdf/epigenetic-regulation-of-the-glucocorticoid-receptor-in-42c0o5px64.pdf

Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse

The genomes of many animals, plants and fungi are tagged by methylation of DNA cytosine. To understand the biological significance of this epigenetic mark it is essential to know where in the genome it is located. New techniques are making it easier to map DNA methylation patterns on a large scale and the results have already provided surprises. In particular, the conventional view that DNA methylation functions predominantly to irreversibly silence transcription is being challenged. Not only is promoter methylation often highly dynamic during development, but many organisms also seem to target DNA methylation specifically to the bodies of active genes.

https://birdlab.bio.ed.ac.uk/bird/sites/sbsweb2.bio.ed.ac.uk.bird/files/20.pdf

DNA methylation landscapes: provocative insights from epigenomics

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.

/pdf/genome-scale-dna-methylation-maps-of-pluripotent-and-2r48wp8zg9.pdf

Genome-scale DNA methylation maps of pluripotent and differentiated cells

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

https://www.pnas.org/content/pnas/89/5/1827.full.pdf

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

The detection of 5'-methylcytosine by the bisulphite-mediated genomic sequencing method has considerably aided study of the role of methylation in areas such as X chromosome inactivation, genomic imprinting and cancer research. However on occasion difficulty has been experienced in obtaining complete conversion of cytosine to uracil in regions of the target DNA. We report here a simple improvement to the method involving addition of urea to the bisulphite reaction, a step which greatly improves the reaction efficiency, presumably by maintaining the target DNA in single stranded form, thereby allowing complete and reliable conversion.

/pdf/urea-improves-efficiency-of-bisulphite-mediated-sequencing-4fgwe87kqn.pdf

Urea improves efficiency of bisulphite-mediated sequencing of 5′-methylcytosine in genomic DNA

To analyse the biological role of 5-methylation of cytosine residues in DNA requires precise and efficient methods for detecting individual 5-methylcytosines (5-MeCs) in genomic DNA. The methods developed over the past decade rely on either differential enzymatic or chemical cleavage of DNA, or more recently on differential sensitivity to chemical conversion of one base to another. The most commonly used methods for studying the methylation profile of DNA, including the bisulphite base-conversion method, are reviewed.

Sequencing 5-methylcytosine residues in genomic DNA.

Breakage of double-stranded DNA due to single-stranded nicking.

A method for detecting presence of a target DNA in a sample, the method comprising: (a) treating a sample containing DNA with an agent that modifies unmethylated cytosine; (b) providing to the treated sample a detector ligand capable of binding to a target region of DNA and allowing sufficient time for a detector ligand to bind to a target DNA; and (c) measuring binding of the detector ligand to DNA in the sample to determine the presence of the target DNA in a sample.

Geoffrey W. Grigg

Papers

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

Urea improves efficiency of bisulphite-mediated sequencing of 5′-methylcytosine in genomic DNA

Sequencing 5-methylcytosine residues in genomic DNA.

Breakage of double-stranded DNA due to single-stranded nicking.

Detection of methylated dna molecules