Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

Abstract

T he potential importance of DNA methylation for specifying epigenetic inheritance in eukaryotic cells was recognized soon after the discovery of the role that methylation plays in the modification and restriction of bacterial and bacteriophage DNA (1-5). In eukaryotic cells, inheritance of the methylated state usually involves 5-methylcytosine and predominantly depends on enzymatic recognition of CpG and CNG motifs. Base-pairing rules (6) ensure that these motifs are symmetrically located on complementary strands of DNA (for example, CpG͞CpG dyads), thus providing the opportunity for the inheritance of cytosine methylation after DNA replication (7). In mammals, maintaining a methylated state of CpG cytosines is an important component of X-chromosome inactivation and genomic imprinting (8-10). The failure to maintain a methylated or an unmethylated state of key cytosines can lead to ''epimutations''; such changes may alter cell and developmental pathways, resulting in new phenotypes (11-14) including disease (15-17). The mechanisms and fidelity of epigenetic inheritance are thus of crucial biological and medical importance.A central issue in epigenetics concerns the mechanism by which a locus maintains a stable epigenetic state through many cell divisions. It appears that epigenetic mechanisms that use 5-methylcytosine within CpG dinucleotides have moderate to high fidelities of maintaining a methylated state of cytosine, after a transitory hemimethylation state during DNA replication (9, 18-23). Hemimethylated sites are also transitional states in developmental processes; active demethylation or de novo methylation may sometimes be involved in gene reactivation or inactivation (24-26). In a study to assess the dynamics of DNA methylation, Riggs and colleagues (9, 27), estimated the fidelity of maintenance methylation (E m ) within partially methylated CpG islands to be Ͼ0.99 per methylated cytosine per cell division; de novo methylation efficiency (E d ) for unmethylated cytosines was estimated to be 0.05 per site per generation. This study, carried out with clones of tissue-culture cells in which methylation was perturbed with 5-azacytidine, also provides a useful mathematical model of the kinetics of DNA methylation (9).Current inferences on epigenetic fidelities and transitional methylation states are based on data for single methylation sites or on patterns of methylation derived from populations of complementary strands. A major experimental limitation has been the difficulty in obtaining methylation patterns from the two complementary strands of an individual DNA molecule. If such a method were available, patterns of methylation fidelity, and detection of both gain and loss of methylation, could be studied relatively directly.We have developed ''hairpin-bisulfite PCR'' for this purpose of analyzing patterns of cytosine methylation on complementary strands of individual DNA molecules. This method uses a hairpin linker, targeted and ligated to restriction-enzyme-cleaved genomic DNA, to maintain attachment o...

The evidence for functional non-CpG methylation in mammalian cells

“…Alternatively, hairpin-bisulphite PCR can identify sites of 5-methylcytosine on complementary strands of restriction enzyme-fragmented DNA by linking them with a hairpin oligonucleotide prior to bisulphite conversion. 27 This method has successfully been used to investigate the symmetry of CpG and non-CpG methylation in mESCs. 28 Detection of non-CpG methylation following PCR of bisulphite converted DNA and sequencing is possible, but requires careful design of primer sequences.…”

Harnessing the developmental potential of nucellar cells: barriers and opportunities

“…In addition, noninvasive techniques for tracking the development of individual nucellar cells in vivo will be needed to identify the precise molecular mechanisms involved. A hairpin-bisulfite polymerase chain reaction method that can detect methylation symmetry between complementary strands of individual DNA molecules [6], if suitably modified for plant tissues, could illuminate the situation. Monitoring single-cell transcription profiles by digital fluorescence microscopy, whereby the nucleus of each cell is tracked by oligonucleotide probes that colourcode the transcription profiles [18], also holds enormous promise in this regard.…”

“…In eukaryotes, epigenetic regulation often involves the methylation of DNA, particularly that of cytosine residues. Failure to transmit faithfully a methylated or unmethylated state (locus) of cytosine can lead to altered phenotypes in plants and animals [6]. Theise and Wilmut [7] point out that, during animal development, cytosine residues of the newly formed DNA strands can be left unmethylated by a passive process for physiological reasons.…”

DNA Methylation in Cancer Tissues

“…Few cancer DNAs digest with Hhlal showed two fractions of NBL2 sequence one with hyper-methylation and one with hypo-methylation. There is evidence that hypo-methylation at NBL2 and hyper-methylation at NBL2 predominates in cancerous cells that suggest site specificity of methylation stats of CpG sites [6,90]. Thus, DNA can be made unstable during carcinogenesis so that CpG sites that are close to each other undergoes opposite changes in methylation D4Z4 (a macro satellite located at sub telomeric region) also exhibit strong hypo-methylation and hyper-methylation in the bulk of array [7].…”