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...

No abstract

DNA methylation within the promoter region of human LINE1 (L1) transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition. Determining methylation patterns on the complementary strands of repeated sequences is difficult using standard bisulfite methylation analysis. Evolutionary changes in each repeat and the variations between cells or alleles of the same repeat lead to a heterogeneous population of sequences. Potential sequence biases can arise during analyses that are different for the converted sense and antisense strands. These problems can be avoided with hairpin-bisulfite PCR, a double-stranded PCR method in which complementary strands of individual molecules are attached by a hairpin linker ligated to genomic DNA. Using human L1 elements to study methylation of repeated sequences, (i) we distinguish valid L1 sequences from redundant and contaminant sequences by applying the powerful new method of molecular barcodes, (ii) we resolve a controversy on the level of hemimethylation of L1 sequences in fetal fibroblasts in favor of relatively little hemimethylation, (iii) we report that human L1 sequences in different cell types also have primarily concordant CpG methylation patterns on complementary strands, and (iv) we provide evidence that non-CpG cytosines within the regions analyzed are rarely methylated. Methylation of CpG dyads within the promoter region of human LINE1 (L1)1 transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition (1-3). Identifying the various states of cytosine methylation in human L1 promoter sequences is thus potentially useful in understanding L1 stability. Methylation states of CpG/CpG dyads are expected usually to be concordant, i.e. fully methylated or fully unmethylated. Methylated cytosines on the parent strand usually give rise to methylated cytosines on the daughter strand, and unmethylated cytosines on the parent strand usually give rise to unmethylated cytosines on the daughter strand because of the properties of DNA methyltransferases during DNA replication (4 -6). CpG dyads that are discordant in methylation, i.e. hemimethylated, are expected to occur at low to moderate frequencies (5, 7-9) with the exception of regions undergoing transcriptional silencing or reactivation and transiently during DNA replication (10 -13). Contrary to this expectation, L1 sequences in human fetal fibroblast cultures were reported to be much more methylated on the sense strand than on the antisense strand (11), which would imply high levels of hemimethylation. Estimating methylation patterns on the complementary strands of repeated sequences can, however, be difficult using conventional bisulfite methylation analysis because few if any of the sampled sense and antisense strand sequences are likely to be derived from the same repeat in the same cell. Even sequences that have an apparent match are likely to be derived from different loci that either had identical sequence in the r...