EP1379685A2 - Nouveau dosage permettant de detecter et de quantifier la semi-methylation - Google Patents

Nouveau dosage permettant de detecter et de quantifier la semi-methylation

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EP1379685A2
EP1379685A2 EP01984994A EP01984994A EP1379685A2 EP 1379685 A2 EP1379685 A2 EP 1379685A2 EP 01984994 A EP01984994 A EP 01984994A EP 01984994 A EP01984994 A EP 01984994A EP 1379685 A2 EP1379685 A2 EP 1379685A2
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methylation
dna
cpg
methylated
cells
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Peter A. Jones
Gangning Liang
Yashitaka Tomigahara
Peter W. Laird
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University of Southern California USC
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University of Southern California USC
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • Patent Application Serial Number 60/247,191 entitled “A NEW ASSAY FOR THE DETECTION AND QUANTITATION OF HEMIMETHYLATION,” filed on 08 November 2000.
  • the present invention relates to DNA methylation, and in particular hemi- methylation.
  • the present invention provides a method to measure the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a particular DNA sequence, in a pool of DNA molecules having mixed DNA methylation states at said CpG dinucleotide.
  • the present invention also provides for methods to calculate the rates of de novo-, and maintenance-methylation per cell division that occur at said CpG dinucleotide.
  • DNA is methylated only at cytosines located 5' to guanosine in the CpG dinucleotide. This modification has important regulatory effects on gene expression predominantly when it involves CpG rich areas (CpG islands) located in the promoter region of a gene sequence. Extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X chromosome of females. Aberrant methylation of normally unmethylated CpG islands has been described as a frequent event in immortalized and transformed cells and has been frequently associated with transcriptional inactivation of tumor suppressor genes in human cancers.
  • DNA methylases transfer methyl groups from a universal methyl donor, such as S- adenosyl methionine, to specific sites on the DNA.
  • Mammalian cells possess methylases that methylate cytosine residues on DNA that are 5' neighbors of guanine in CpG dinucleotides (CpG) . This methylation may play a role in gene inactivation, cell differentiation, tumorigenesis, X-chromosome inactivation, and genomic imprinting. CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation and parental specific imprinting where methylation of 5' regulatory regions can lead to transcriptional repression.
  • CpG dinucleotides CpG dinucleotides
  • DNA methylation is also a mechanism for modifying the base sequence of DNA without altering its coding function.
  • DNA methylation is a heritable, reversible and epigenetic change. Yet, DNA methylation has the potential to alter gene expression, which has profound developmental and genetic consequences.
  • DNA methylation is required for mammalian development (Li et al, 1992; Okano et al., 1999) yet the mechanisms responsible for the establishment and copying of methylation patterns remain almost completely unknown.
  • 5-Methylcytosine is asymmetrically distributed in the genome and is most commonly found in CpG-poor regions, since most CpG islands in somatic cells remain methylation-free, except for the promoters of imprinted genes and genes on the inactive X-chromosome (Bird et al., 1985).
  • Dnmtl The predominant Dnmt in the cell, Dnmtl, was cloned and characterized by Bestor and colleagues (1988) and is localized to replication machines in the S-phase nucleus (Leonhardt et al., 1992; Rountree et al., 2000). Since Dnmtl shows a preference for hemimethylated CpG pairs (Gruenbaum et al., 1981; Bestor and Ingram, 1983), it is considered to be an excellent candidate for copying the pattern of methylation present on the parental strand after DNA has been replicated. However, Dnmtl is capable of modifying unmethylated DNA in the test tube, and is thus also a candidate for inducing de novo methylation.
  • satellite DNAs appear to be a preferred target for the human DNMT3B enzyme, because these satellite DNA sequences are specifically undermethylated in patients with ICF syndrome, characterized by germ-line mutations in the DNMT3B gene (Hansen et al., 1999; Okano et al., 1999; Xu et al., 1999).
  • analysis of individual cloned PCR products for the presence of C or T at the first position of the specific CpG dinucleotide was accomplished using a methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay (Gonzalgo and Jones, 1997).
  • Ms-SNuPE methylation-sensitive single nucleotide primer extension
  • the present invention further provides a hemimethylation detection kit useful for measuring the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a pool of DNA with mixed DNA methylation states at that CpG.
  • the present invention further provides a hemimethylation detection kit useful for measuring the rate of de novo methylation (n) and the rate of maintenance methylation (m) at a single CpG dinucleotide within a palindromic CpG methylation site.
  • Rsal refers to the art-recognized restriction endonuclease having the "GTAC” (top strand): “CATG” (bottom strand) recognition motif.
  • MSP Metal-specific PCR
  • Genomic DNA was obtained following lysis with 100 mM NaCl, 10 mM ⁇ DTA, 1% SDS, and 1 ⁇ g/ml Proteinase K and purified by phenol and chloroform extractions and ethanol precipitation. The yield and purity of the DNA was determined by optical density measurement and 300 ⁇ g of DNA digested with 3000 units of Rsal (Roche) for 16 hr at 37°C. Digested DNA was purified again by phenol and chloroform extractions, ethanol precipitated, and resuspended in 500 ⁇ l T ⁇ buffer pH 7.5 with 50 ⁇ l 10X immunoprecipitation buffer (sodium phosphate (pH 7.0)), 0.14 M NaCl, 0.05% Triton X- 100).
  • the precipitate was collected after 5 min centrifugation at 13,000 rpm in a cold microcentrifuge, dissolved in 200 ⁇ l T ⁇ pH 7.5 and treated with Proteinase K at 50°C for more than 12 hr. DNA was deproteinized by phenol and chloroform extractions and ethanol precipitated.
  • Typical reagents for Ms-SNuPE analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE primers for specific gene; reaction buffer (for the Ms-SNuPE reaction); and radioactive nucleotides.
  • bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
  • Methylation analysis was performed using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay (Gonzalgo and Jones, 1997). For Examples 1-5. below:
  • Ms-SNuPE primers for top strand site 2 (5'-AATAATTTTGTTTTTTTGGATATT- 3') (SEQ ID NO:5), site 3 (5'-AAATTTTGTTTTTGGTTGTAAA-3') (SEQ ID NO:6), for bottom strand site 2 (5'-AGGAATAGAATTTGAGATATT-3') (SEQ ID NO:7), site 3 (5'- TAAATTGTTTTAATTAGATTAATAA-3 ') (SEQ ID NO:8).
  • Ms-SNuPE reactions were performed in 10 ⁇ l total volume under the following conditions: 4 ⁇ l Qiaquick product, 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2s 100 mM KCl, 0.5 ⁇ M final concentration of each primer, and 1 ⁇ Ci of either [ 32 P]dCTP or [ 32 P]dTTP.
  • Primer extension conditions were: 95°C for 2 min, 50°C for 2 min, 72°C 1 min. The reactions were combined with 4 ⁇ l stop solution before being denatured at 95°C for 5 min and loaded onto a 15% denaturing polyacrylamide gel (7M urea).
  • PCR primers were designed specifically to amplify only bisulfite converted DNA and control experiments showed non-amplification of unconverted DNA with these primers. Also, sequencing of PCR products after bisulfite treatment showed less than 1% residual Cs at not CpG sites indicating that the assays were valid for methylation status at CpG sites.
  • PCR products were gel purified with the Qiaquick Gel Extraction Kit (Qiagen), and the template was resuspended in 30 ⁇ l H 2 O. Ms-SNuPE Primers.
  • Ms-SNuPE reactions were performed in 10 ⁇ l total volume under the following conditions: 4 ⁇ l Qiaquick product, 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2 , 100 mM KCl, 0.5 ⁇ M final concentration of each primer, and 1 ⁇ Ci of either [ 32 P]dCTP or [ 32 P]dTTP.
  • Primer extension conditions of Cl-f and CII-d were: 95°C for 1 min, 46°C for 30 sec, 72°C for 20 sec.
  • the reactions were combined with 4 ⁇ l stop solution before being denatured at 95°C for 5 min and loaded onto a 15% denaturing polyacrylamide gel (7M urea). Quantitation of methylation levels was performed on a Molecular Dynamics Phosphorlmager.
  • Bisulfite genomic sequencing by Ms-SnuPE Bisulfite Genomic Sequencing by Ms-SNuPE or Automated DNA Sequencer. Traditionally patterns are determined by the cloning of individual bisulfite treated molecules followed by DNA sequencing. It was more convenient to subject individually cloned molecules to Ms-SNuPE analysis to rapidly assess how the four sites were methylated (Cl-f and CII-d). The PCR products from bisulfite converted DNA were ligated into pCRII cloning vector (Invitrogen, San Diego, CA). Individual plasmid clones were amplified by Ml 3 primers (forward and backward). The PCR product was used to sequence by Ms-SNuPE.
  • Hemimethylation assay The fact that the enzyme Hpall will not cut the sequence CCGG in either the fully or hemimethylated configuration followed by bisulfite treatment was used to determine whether unmethylated cytosine occurred in the context of a CpG sequence in a Hpall insensitive site.
  • Two ⁇ 4 ⁇ g D ⁇ A were digested by Rsal and Hpall (10 units per ⁇ g) for 16 hr at 37°C and then Hpall added (10 units per ⁇ g) for another 2 hr. The digests were then analyzed for methylation at site 2 by cloning of individual PCR-amplified bisulfite treated molecules as described above.
  • Methylation-Sensitive AP-PCR Methylation-sensitive AP-PCR and isolation of fragments of interest were performed as previously described Gonzalgo, et al., Cancer Research 57:594-599, 1997; Liang, et al., Genomics 53:260-268, 1998).
  • the following CpG- poor primers were used for AP-PCR analysis: GCP1, 5'-CACATGGTTCTGC-3' (SEQ ID NO:13); GCP2, 5'-GTCTCTATGACCC-3' (SEQ ID NO:14); GCP4, 5'- CTTACTGTGCCAC-3' (SEQ ID NO: 15).
  • the following pairs were used in the AP-PCR reaction: GCP1/GCP2, GCP2/GCP4, GCP1/GCP4.
  • Methylation Assay Procedures Various methylation assay procedures are known in the art, and can be used in conjunction with the present invention for sequence determinations. These assays allow for determination of the methylation state of one or a plurality of CpG dinucleotides (e.g., CpG islands) within a DNA sequence. Such assays involve, among other techniques, DNA sequencing of bisulfite-treated DNA, PCR (for sequence-specific amplification), Southern blot analysis, use of methylation-sensitive restriction enzymes, etc.
  • DNA methylation levels at specific gene loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite- treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by
  • Typical reagents for COBRA analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); restriction enzyme and appropriate buffer; gene-hybridization oligo; control hybridization oligo; kinase labeling kit for oligo probe; and radioactive nucleotides.
  • bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
  • assays such as "MethyLight” (a fluorescence-based real-time PCR technique) (Eads et al, Cancer Res. 59:2302-2306, 1999), Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) reactions (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP”; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; US Patent No. 5,786,146), and methylated CpG island amplification ("MCA”;Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with other of these methods.
  • MSP methylation-specific PCR
  • MCA methylated CpG island amplification
  • the MethyLight may assay be used as a quantitative test for methylation patterns in the genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization.
  • the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site.
  • An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides.
  • the MethyLight process can by used with a "TaqMan®” probe in the amplification process.
  • double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using TaqMan® probes; e.g., with either biased primers and TaqMan® probe, or unbiased primers and TaqMan® probe.
  • the TaqMan® probe is dual-labeled with fluorescent "reporter” and "quencher” molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about 10 °C higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing/extension step.
  • Taq polymerase As the ⁇ Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5' to 3' endonuclease activity will then displace the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system.
  • Typical reagents for MethyLight analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); TaqMan® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.
  • Typical reagents for Ms-SNuPE analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE primers for specific gene; reaction buffer (for the Ms-SNuPE reaction); and radioactive nucleotides.
  • bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
  • MSP methylation-specific PCR
  • DNA is modified by sodium bisulfite converting all unmethylated, but not methylated cytosines to uracil, and subsequently amplified 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.
  • Typical reagents e.g., as might be found in a typical MSP-based kit
  • MCA MCA.
  • the MCA technique is a method that can be used to screen for altered methylation patterns in genomic DNA, and to isolate specific sequences associated with these changes (Toyota et al., Cancer Res.
  • Typical reagents for MCA analysis may include, but are not limited to: PCR primers for arbitrary priming Genomic DNA; PCR buffers and nucleotides, restriction enzymes and appropriate buffers; gene-hybridization oligos or probes; control hybridization oligos or probes.
  • kits for Detection of Hemimethylated CpG-containing Nucleic Acid provides a hemimethylation detection kit for measuring the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a pool of DNA with mixed DNA methylation states at that CpG.
  • the present invention further provides a hemimethylation detection kit useful for measuring the rate of de novo methylation (n) and the rate of maintenance methylation (m) at a single CpG dinucleotide within a Hpall site.
  • the reagents required to perform one or more art-recognized methylation assays are combined with specific primers or probes to determine the hemimethylation state of CpG-containing nucleic acids, or the rate of de novo methylation ( ⁇ ) and the rate of maintenance methylation (m) at a single CpG dinucleotide within a Hpall site, according to the present invention.
  • the Ms-SnuPE methylation assay could be used alone or in combination with other methylation assay methods known in the art, along with specific primers or probes to determine the hemimethylation state of a CpG dinucleotide within a particular genomic sequence.
  • the methylation status of the four CpG sites in individual DNA strands in cells containing all three Dnmts (M1/3A/3B cells) or containing Dnmtl (Ml cells) or Dnmt3a and 3b only (M3A/3B cells) were assessed in detail to investigate potential differences in the patterns of methylation.
  • the nomenclature used to describe the cell types was selected to focus on which enzymes were present in the cells rather than those which > were absent. Minimal differences in methylation levels and patterns were seen in cells lacking either Dnmt 3 a or 3b only. Initially, the methylation patterns at sites 2-4 were determined by cloning of individual bisulfite treated molecules followed by DNA sequencing (top fragments in Fig. IB).
  • Figure 1 A shows the patterns of methylation in ES Cells.
  • the methylation status of the four CpG sites in individual molecules of DNA (Fragment Cl-f) comprising the 440 bp sequence were assessed by cloning of individual molecules followed by MS-SNuPE analysis of the four sites as indicated.
  • Example gels of samples labeled with either dCTP "C” or dTTP "T" are shown.
  • the data from the upper section within each panel were obtained by direct sequencing of fragments containing sites 2 through 4.
  • the bottom sections are for all four sites.
  • the status of methylation at the sites in individual molecules is read from left to right.
  • Figure IB also shows the average percent of methylation at the four sites in the three cell types examined.
  • Sites 2 and 4 were most methylated on average whereas sites 1 and 3 were less efficient methyl acceptors.
  • the methylation pattern in the M3A/3B cells were, however, completely different in that two classes of molecules were present. The majority of molecules in which all four sites were simultaneously assessed were methylation-free (72%), 14% had only one site methylated but 14% had multiple sites methylated on a single molecule.
  • Figure 2 shows the detection and quantitation of hemi-methylation in individual cloned DNA molecules according to the present invention.
  • the experimental approach used to detect hemi-methylation in the ES cells comprises precutting genomic DNA with Hpall, followed by bisulfite treatment, then cloning individual PCR products, and assessing the methylation status of, e.g., site 2 by MS-SNuPE analysis. Typical results are shown for these MS-SNuPE results in which a signal in the T lanes of example clones indicates hemimethylation with the top strand being unmethylated.
  • the presence of a signal in the "C" lane indicates that the site may be either hemi-methylated (i.e., the bottom strand is unmethylated) or fully methylated.
  • the distribution between these two scenarios is determined by calculation, assuming that there is an equal probability of hemi-methylation of either the top or bottom strand.
  • DNA extracted from the various cell types was digested with an excess of Hpall, bisulfite treated and then amplified by PCR with primers flanking the Hpall site (site 2). Since the Hpall site is located between the primers, molecules without full or hemi- methylation of site 2 would not be amplified and thus be excluded from the analysis.
  • the degree of hemi-methylation at a particular site can be accurately determined by averaging results obtained by analysis of individual cloned PCR products for the presence of C or T at site 2 (for example). Since only the top strand is analyzed in this approach and methylation of either the top or bottom strand is possible, the degree of hemi-methylation was calculated as indicated in "Experimental Procedures," above. These calculations assume that there is an equal probability of hemi-methylation of either the top or bottom strand, a supposition which was supported by additional experiments (see Examples 6-8 below).
  • Figure 3 shows the measured steady state methylation levels, methylation rates, and an overview of the procedure used to calculate de novo (n) and maintenance (m) methylation rates (expressed as the fraction of substrate molecules converted per cell division) at a single CpG dinucleotide within a Hpall site, starting with the measurement of two experimental variables ("P" and "S").
  • P two experimental variables
  • S two experimental variables
  • the unmethylated CpGs are absent in the case of the S and D variables, since the Hpall digestion removes these molecules from consideration at this step.
  • the data obtained for the three cell lines for the measured variables "P" and "S” are shown in the columns on the right as percentages, followed by the absolute numbers of molecules assessed. It is assumed that the top and bottom strand are equal in their methylation levels and rates. On average, differences between the newly synthesized strand and daughter strand after DNA replication should be distributed equally between the top and bottom strand in a large population of cells.
  • Figure 3 also shows the rates of de novo and maintenance methylation which could be calculated from this data (see “Experimental Procedures,” above). Interestingly, Figure 3 shows that M3A/3B cells had higher rates of hemi-methylase activity (0.37) than of de novo methyltransferase activity (0.07). These results indicate that Dnmtl and Dnmt3a/3b acted coordinately to ensure a low proportion of hemi-methylated sites in the wild type cells.
  • Figure 4A summarizes results from such pulse-chase experiments which show that DNA synthesized during a 1 hr pulse of BrdU in wild type M1/3A/3B cells was not methylated to its final level until 24-48 hr after synthesis.
  • ES cells containing the genes for the indicated DNA methyltransferases were pulsed for 1 hr with BrdU. The pulse was then removed and fresh medium added to the cells during the chase period. The DNA was extracted from the cells at various times after the pulse began and immunoprecipitated to isolate the BrdU containing DNA. The methylation status of sites 2 and 3 were averaged at the indicated time points after determination by quantitative MS-SNuPE analysis. The data represent the mean values for the determinations on the top and bottom strands in two to three experiments and the error bars indicate the standard deviations.
  • FIG 4B also shows that the maturation process was not blocked by drugs which inhibit histone deacetylase (Trichostatin A, "TSA”), DNA synthesis (Aphidicolin) or mitosis (Colcemid).
  • TSA histone deacetylase
  • Aphidicolin DNA synthesis
  • Colcemid mitosis
  • DNA collected from these two time points was immunoprecipitated to isolate BrdU-containing DNA, and the methylation status of individual molecules determined to assess the methylation density of these molecules as a function of time after synthesis as in Figure 1. Data are given as the percentage of individual molecules containing between 0 and 4 sites concurrently methylated on an individual molecule.
  • DNA collected immediately after the 1 hr pulse from M1/3A 3B cells showed that a considerable proportion (38%) of molecules contained none or only one of the four sites methylated and a relatively even distribution of methylation densities.
  • the pattern shifted substantially in the 24 hr chase period with most of the molecules now acquiring concurrent methylation at three or four sites.
  • Newly synthesized DNA in Ml cells had 40% of molecules with zero or one site methylated, 9% having two sites and 50% having three or four sites methylated. Again, this pattern shifted during the chase period, but the mode of the density distribution was two sites methylated per molecule as compared to the three in M1/3A/3B cells.
  • Example 5 Subcellular Localization of DNA Methyltransferases If the discontinuity observed in the kinetics of post-synthetic restoration of DNA methylation levels was indeed attributable to distinct methyltransferase activities, with Dnmtl mainly responsible for maintenance activity immediately following DNA synthesis and Dnmt3a and/or 3b being active later in the cell cycle, then Dnmt3a and/or 3b would not be expected to be as strongly associated with toroidal replication foci, as has been observed for Dnmtl.
  • the subcellular localization of the three methyltransferases was determined by the transfection of plasmids containing the three genes fused to green fluorescence protein (GFP) into mouse C3H 10T1/2 C18 cells. Cells were double stained with an antibody to PCNA labeled with Texas red and examined by confocal microscopy 48-72 hr after transfection. Cells transfected with DNMT-1 confirmed earlier studies that this protein often co-localized with PCNA, particularly in toroidal structures presumably representing replication machines at late S phase (Leonhardt et al., 1992; Chuang et al., 1997; Rountree et al, 2000).
  • Dnmt3a and 3b were localized almost entirely to the nucleus and were more diffusely present in nuclei containing these toroids.
  • Dnmt3b unlike Dnmt3a showed some co-localization with the PCNA-containing toroids.
  • Dnmt3b but not Dnmt3a may associate with replication machines.
  • EXAMPLE 6 Genetic scanning was used to identify sequences to investigate methylation patterns. The findings of Examples 1-5 (above) were extended. Specifically, a genome scanning approach was used to investigate the patterns of methylation in the various knockout ES cells in CpG poor and CpG rich regions to determine the roles of the enzymes in carrying out the bulk of methylation in mouse embryonic stem (ES) cells.
  • ES mouse embryonic stem
  • methylation levels of CpG-poor sequences were, in general, uniformly reduced in Dmntl -deficient cells. However, there was considerable variability among different regions in the efficiency with which DNA methylation was retained in Dnmt3a/3b-deficient cells indicating a sequence preference of the Dnmtl enzyme.
  • MS AP-PCR was used to fingerprint the methylation patterns in a panel of ES cells containing different combinations of DNA methyltransferases (Dnmtl (-/-) or Dnmt3a (-/- )/3b (-/-)).
  • the MS AP-PCR method allows for a methylation pattern to be easily obtained and relies on the differential susceptibilities of unmethylated and methylated CCGG sites to cutting by the enzyme Hpall giving a valid fingerprint of the methylation status of CpG islands.
  • the purpose and advantage of Ms AP-PCR was to perform a rapid and global screen of genome which would then allow identification of representative sequences for more detailed analysis. We initially focused on CpG poor regions of DNA in the current work, since these are the regions of DNA in which the majority of 5-methylcytosine is found.
  • Figure 7A shows an example of an analysis of DNA extracted from the various cell types using CpG poor primers to target regions of DNA not located in CpG islands.
  • the nomenclature used for the cells was selected to highlight which gene products were active in the cells rather than those which were absent.
  • Analysis of the fingerprints showed a uniform loss of methylation at most evaluable bands in cells which contained Dnmt3a and 3b only (M3A/3B), as compared to the other cell types examined (see lanes 2 and bands indicated by filled in arrows).
  • Class I sequences as those that had decreased methylation in M3A/3B cells (i.e., lacking Dnmtl), but close to normal methylation levels in Ml cells.
  • Class II sequences on the other hand showed loss of methylation in both Ml and M3A/3B cells.
  • Figure 8 shows the sequence properties of a selection of these two classes of fragments with respect to the occurrence of the CpG dinucleotides and the presence of repetitive elements.
  • Class I sequences tended to have fewer repetitive elements, and a slightly lower CpG density than Class II sequences.
  • this distinction was not absolute since there were no obvious differences with respect to these properties such as the fragments Cl-b and Cll-a, or Cl-a and Cll-b.
  • Dnmt3a and 3b which appear to be processive enzymes whose activities fill in the patches in addition to their roles as de novo methylases.
  • the present invention provides novel methods to measure hemi-methylation and to calculate methylation rates. These novel methods were used herein to conclude that Dnmt3a and 3b act mainly as maintenance methyltransferases to ensure the completeness of methylation of the genome in ES cells.
  • the method is robust and has general utility in other experimental and applied applications.
  • the occurrence of a high percentage of hemimethylated sites in the M3 A/3B cells seemed, at first sight, to not fit with the suggestion that the Dmnt3a and 3b enzymes acted as hemi-methylases to fill in gaps in wild-type cells.
  • This apparent paradox could be explained if the overall level of cytosine methyltransferase activity in the M3A/3B cells was inadequate for effective methylation maintenance.

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Abstract

La présente invention concerne un procédé qui permet de mesurer la fraction de molécules d'ADN qui est semi-méthylée au niveau d'un dinucléotide CpG spécifique, dans une séquence d'ADN particulière, dans un groupe de molécules d'ADN ayant des états de méthylation d'ADN mélangés au niveau dudit dinucléotide CpG; des procédés de calcul des taux de méthylation de novo et d'entretien par division cellulaire qui se produisent au niveau dudit dinucléotide CpG. Cette invention concerne également des trousses de méthylation destinées à être utilisées pour mesurer et quantifier la semi-méthylation et pour calculer les taux de méthylation de novo et d'entretien.
EP01984994A 2000-11-08 2001-11-08 Nouveau dosage permettant de detecter et de quantifier la semi-methylation Withdrawn EP1379685A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US24719100P 2000-11-08 2000-11-08
US247191P 2000-11-08
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US20060134643A1 (en) 2000-06-19 2006-06-22 Kurt Berlin Bisulfite conversion of DNA
DE10347399B4 (de) * 2003-10-09 2005-09-15 Epigenomics Ag Bisulfit-Umwandlung zum Nachweis von Cytosin-Methylierungen in DNA mittels optimierter Aufreinigung
EP1339868A2 (fr) 2000-06-23 2003-09-03 The University of Chicago Methodes d'isolation d'adn centromere
DE10346363B4 (de) * 2003-09-30 2005-09-29 Epigenomics Ag Verfahren zur Methylierungsanalyse von DNA
CA2540310A1 (fr) * 2003-10-09 2005-04-28 Epigenomics Ag Transformation amelioree de bisulfite d'adn
WO2005040399A2 (fr) * 2003-10-21 2005-05-06 Orion Genomics Llc Procedes pour quantifier la densite de methylation d'un site d'adn
DE102005011398A1 (de) * 2005-03-03 2006-09-14 Epigenomics Ag Verfahren zur Untersuchung von Cytosin-Methylierungen in DNA

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US6017704A (en) * 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US6251594B1 (en) * 1997-06-09 2001-06-26 Usc/Norris Comprehensive Cancer Ctr. Cancer diagnostic method based upon DNA methylation differences

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