Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts.

We report here a new, sensitive and versatile genomic sequencing method, which can be used for in vivo footprinting and studies of DNA adducts. Starting with mammalian genomic DNA, single-stranded products are made by repeated primer extension; these products are subjected to homopolymeric ribonucleotide tailing at the 3' termini with terminal deoxynucleotidyl transferase and then ligated to a double-stranded linker having a complementary 3' overhang, and used for PCR. This terminal transferase-dependent PCR (TDPCR) method can generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR). A UV photofootprint in the mouse Xist gene promoter can be easily detected using TDPCR. No special enzymes or chemical reagents are needed to convert DNA adducts into strand breaks. Any lesion that blocks primer extension should be detectable.     

Characterization of a pollen-specific cDNA from sunflower encoding a zinc finger protein.

The maize transposable element Activator (Ac) carries subterminal CpG-rich sequences which are essential for the transposition of the element. It has previously been shown that the methylation of certain sequences contained in this region can alter their ability to interact with the Ac-encoded protein. The novel hypothesis that the methylation of subterminal Ac sequences is required for transposition was tested. Approximately 150 bp of the 5' subterminal region of the Ac element was examined for the presence of 5-methylcytosines by the ligation-mediated polymerase chain reaction (LMPCR)-aided genomic sequencing method. The methylation status of 22 and 39 cytosines on either strand of the DNA were analysed in each of five different transgenic tobacco cultures carrying transposable Ac sequences. Ten micrograms of tobacco DNA were used for each base-specific cleavage reaction before amplification by LMPCR. All but one of the cytosines were unmethylated. Only a minor fraction of the Ac molecules was methylated at one cytosine residue. It is concluded that DNA methylation at the tested Ac sequences is not required for the transposability of Ac or Ds elements in tobacco cells.     

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.     

LMPCR for detection of oligonucleotide-directed triple helix formation: a cautionary note.

We note that precautions are necessary when ligation-mediated PCR (LMPCR) is applied to the detection of oligonucleotide-directed triple helix formation in vitro and in vivo. Synthetic oligonucleotides applied to cell cultures can persist after chemical treatment and genomic DNA isolation and inhibit a key step in LMPCR, causing an artifact that simulates a triplex footprint. Residual oligonucleotides apparently form triplexes during LMPCR, blocking ligation of the unidirectional linker in a site-specific manner. We show that careful removal of residual oligonucleotide prior to LMPCR alleviates this problem.     

Optimal conditions to use Pfu exo– DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA–protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo^– DNA polymerase (Pfu exo^–). The relative efficiency of Pfu exo^– was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo^– proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo^–, while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo^– was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo^– at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

DNA methylation is an indispensible epigenetic modification required for regulating the expression of mammalian genomes. Immunoprecipitation-based methods for DNA methylome analysis are rapidly shifting the bottleneck in this field from data generation to data analysis, necessitating the development of better analytical tools. In particular, an inability to estimate absolute methylation levels remains a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling. To address this issue, we developed a cross-platform algorithm-Bayesian tool for methylation analysis (Batman)-for analyzing methylated DNA immunoprecipitation (MeDIP) profiles generated using oligonucleotide arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). We developed the latter approach to provide a high-resolution whole-genome DNA methylation profile (DNA methylome) of a mammalian genome. Strong correlation of our data, obtained using mature human spermatozoa, with those obtained using bisulfite sequencing suggest that combining MeDIP-seq or MeDIP-chip with Batman provides a robust, quantitative and cost-effective functional genomic strategy for elucidating the function of DNA methylation.     

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.     

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.     

一种用于DNA甲基化分析的改进型亚硫酸氢盐转化法

DNA甲基化分析是认识生理、病理条件下基因表达变化的重要途径.亚硫酸氢盐转化是DNA甲基化分析的瓶颈.本文旨在改进琼脂糖 亚硫酸氢盐DNA处理方案(agarose bisulfite method),建立一种简便稳定、适合常规甲基化分析的亚硫酸氢盐转化法.把DNA包入普通琼脂糖,以饱和亚硫酸氢盐在较高的温度下快速处理,然后用离心柱型琼脂糖凝胶DNA回收试剂盒,集DNA凝胶回收、脱盐、脱磺基和纯化于一体,完成整个转化过程.Bisulfite-PCR、克隆测序和酶切法分析转化率、转化特异性和转化物的质量.用该方案处理的HeLa细胞DNA,多个片段的转化率均大于98%,甲基化片段96.2%的CpG保持不变,可以扩增605 bp的较大片段,灵敏度介于普通法和琼脂糖亚硫酸氢盐法之间,而重复性较二者都好.改良后的方案简化了操作流程,快速稳定,易学易用,可实现高效特异转化,适合于一般实验者对常规检材进行DNA甲基化分析.     

High-Speed Conversion of Cytosine to Uracil in Bisulfite Genomic Sequencing Analysis of DNA Methylation

Bisulfite genomic sequencing is a widely used technique foranalyzing cytosine-methylation of DNA. By treating DNA withbisulfite, cytosine residues are deaminated to uracil, whileleaving 5-methylcytosine largely intact. Subsequent PCR andnucleotide sequence analysis permit unequivocal determinationof the methylation status at cytosine residues. A major caveatassociated with the currently practiced procedure is that ittakes 16–20 hr for completion of the conversion of cytosineto uracil. Here we report that a complete deamination of cytosineto uracil can be achieved in shorter periods by using a highlyconcentrated bisulfite solution at an elevated temperature.Time course experiments demonstrated that treating DNA with9 M bisulfite for 20 min at 90°C or 40 min at 70°C allcytosine residues in the DNA were converted to uracil. Underthese conditions, the majority of 5-methylcytosines remainedintact. When a high molecular weight DNA derived from a cellline (containing a number of genes whose methylation statuswas known) was treated with bisulfite under the above conditionsand amplified and sequenced, the results obtained were consistentwith those reported in the literature. Although some degradationof DNA occurred during this process, the amount of treated DNArequired for the amplification was nearly equal to that requiredfor the conventional bisulfite genomic sequencing procedure.The increased speed of DNA methylation analysis with this novelprocedure is expected to advance various aspects of DNA sciences.     

Regulation and function of mammalian DNA methylation patterns: a genomic perspective

Mammalian DNA methyltransferases (DNMTs) establish and maintain genomic DNA methylation patterns that are required for proper epigenetic regulation of gene expression and maintenance of genome stability during normal development. Aberrant DNA methylation patterns are implicated in a variety of pathological conditions including cancer and neurological disorders. Rapid advances in genomic technologies have allowed the generation of high resolution whole-genome views of DNA methylation and DNA methyltransferase occupancy in pluripotent stem cells and differentiated somatic cells. Furthermore, recent identification of oxidation derivatives of cytosine methylation in mammalian DNA raises the possibility that DNA methylation patterns are more dynamic than previously anticipated. Here, we review the recent progress in our understanding of the genomic function and regulatory mechanisms of mammalian DNA methylation.     

DNA methylation dynamics during the mammalian life cycle

DNA methylation is dynamically remodelled during the mammalian life cycle through distinct phases of reprogramming and de novo methylation. These events enable the acquisition of cellular potential followed by the maintenance of lineage-restricted cell identity, respectively, a process that defines the life cycle through successive generations. DNA methylation contributes to the epigenetic regulation of many key developmental processes including genomic imprinting, X-inactivation, genome stability and gene regulation. Emerging sequencing technologies have led to recent insights into the dynamic distribution of DNA methylation during development and the role of this epigenetic mark within distinct genomic contexts, such as at promoters, exons or imprinted control regions. Additionally, there is a better understanding of the mechanistic basis of DNA demethylation during epigenetic reprogramming in primordial germ cells and during pre-implantation development. Here, we discuss our current understanding of the developmental roles and dynamics of this key epigenetic system.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

Genome-Wide Analysis of DNA Methylation Dynamics during Early Human Development

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.     

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.     

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.     

High density DNA methylation array with single CpG site resolution

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R² of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.