The main focus in proteomic studies is on the identification of proteins in given biological samples. Proteins isolated and separated from a given sample (e.g., whole cell lysates, blood or tissue, protein complexes) by immunoprecipitation or affinity chromatography or two-dimensional elect ... more
Analysis of the DNA Methylation Patterns at the BRCA1 CpG IslandFrédérique Magdinier1 and Robert Dante2*
1Laboratory of Molecular Biology of the Cell, Ecole Normale Superieure, Lyon, France
2Laboratory of Molecular Oncology, Centre Léon Bérard, Lyon, France
Germ-line alterations of the BRCA1 gene confer a lifetime risk of 40% for ovarian cancers and of 40%-80% for breast cancers. It is likely that BRCA1 acts as a tumor suppressor gene. BRCA1 involvement in breast cancers does not seem to be restricted to familial cancers. Despite the absence of somatic mutations in the breast tissues, a down regulation of BRCA1 expression is associated with malignancy in human sporadic breast cancers .
In tumor cells, aberrant methylation of CpG dinucleotides at the 5’ end of tumor suppressor genes is frequently associated with gene silencing. However, analysis of the DNA methylation patterns indicated that only a minor fraction (10% to 20%) of breast tumors exhibited methylated CpGs at the promoter region, position –258 to +43 from the transcription start site of BRCA1 . Taken together, these data suggest that additional epigenetic events may be involved in the down regulation of BRCA1 in breast cancers.
The BRCA1 gene spans 81 kb of genomic DNA and shares a bidirectional promoter with NBR2 (next to BRCA1 gene 2; Figure 1). This regulatory region is embedded in a large CpG-rich region of ~2.8 kb in length, from nt –1810 to nt +974 (Figure 1). Since DNA methylation can repress gene transcription at a distance (1 kb–2 kb) from the promoter region, we have investigated the methylation status of this BRCA1 CpG island.
Materials and Methods
DNA was extracted from frozen pulverized tissue samples and cells by the standard proteinase K/phenol/chloroform procedure. A similar method with the addition of 0.001% (v/v) ß-2-mercaptoethanol was used to prepare decondensed DNA from spermatozoa. Human oocytes that failed to fertilize 24 hours after conventional IVF were collected from the Assisted Conception Unit (E. Herriot Hospital, Lyon, France). When DNA was analyzed from oocytes (6–10/assay), 2 µg of plasmid DNA was added as carrier to the samples in a final volume of 100 µl of 50 mM Tris-50 mM EDTA buffer containing 0.25% SDS and 14 µg/ml of proteinase K. The mixture was incubated at 55°C for 2 hours, and then the samples were processed as described in the "bisulfite modification" section.
PCR-based methylation assay
DNA extracted from tissue samples and cell lines was digested with a fivefold excess of restriction enzyme and incubated overnight at 37°C in the appropriate buffer. Enzymes were inactivated by heating at 65°C for 1 hour, and an aliquot (10 ng) of the reaction was used for PCR amplification. The PCR amplification was performed in the following conditions: standard Taq polymerase buffer, 4% DMSO, 3 mM MgCl2, 100 µM of each of the four deoxyribonucleoside triphosphates, 0.25 µM of the primers (forward: 5´-TTGGGAGGGGGCTCGGGCAT-3´; reverse: 5´ CAGAGCTGGCAGCGGACGGT-3´) and 0.6 units of Taq DNA polymerase after 35 cycles in an Eppendorf thermocycler (1 minute denaturation at 94°C, 2 minutes annealing at 55°C and 3 minutes extension at 72°C).
The sodium bisulfite reaction was carried out in 100 µl from 4 µg of DNA (3 µg of carrier DNA and 1 µg of human genomic DNA) or 2 µg of carrier and the oocyte DNA extract. Alkali denatured DNA (0.5 M NaOH, 30 minutes at 37°C) was incubated in 3 M NaHSO3 and 5 mM hydroquinone for 16 hours at 50°C. Modified DNA was purified using a commercially available DNA clean-up system and eluted into 50 µl of sterile water. Modification was completed by 0.3 M NaOH and DNA was precipitated by ethanol in 0.5 M ammonium acetate pH 4.6 and resuspended in water.
DNA was amplified using a nested PCR. The first round of PCR amplification was accomplished in 100 µl in standard Taq polymerase buffer, 100 µM of each of the 4 deoxyribonucleoside triphosphates, 3 mM MgCl2, 0.25 µM of the primers (forward: 5´-TTTTGTTTTGTGTAGGGCGGTT-3´; reverse: 5´-CCTTAACGTCCATTCTAACCGT-3´) and 0.6 units of Taq DNA polymerase in 35 cycles (1 minute denaturation at 94°C, 2 minutes annealing at 55°C and 3 minutes extension at 72°C). An aliquot of the first amplification was reamplified with internal primers (forward: 5´-TGAGAATTTAAGTGGGGTGT-3´; reverse: 5´-AACCCTTCAACCCACCACTAC-3´) under the same conditions.
Results and Discussion
PCR-based methylation assay
In a first set of experiments, we investigated the overall methylation level of the BRCA1 CpG island using a PCR-based methylation assay. In order to normalize the length of genomic DNA fragments, DNA was cleaved with the Rsa I enzyme. Then, samples were digested with Cfo I (GCGC site) and Hpa II (CCGG site) enzymes, which are inhibited by the methylation of the internal cytosine, and as a control, with Msp I (CCGG site) which is insensitive to the methylation of this cytosine. Thus, Rsa I digestion cuts whithin the BRCA1 CpG island fragment (nt –3053 to nt –649) and the -1,714 to -1,005 region were amplified by PCR. In each experiment, the sample digested with Rsa I was amplified to verify the efficiency of the amplification. The sequence analyzed contains 9 Hpa II sites, and 9 Cfo I sites. PCR amplification occurs only when the sites are methylated, and therefore uncut by the two methylation sensititve enzymes. Representative experiments are shown (Figure 1).
Data obtained indicated that Hpa II and Cfo I sites were methylated in human somatic tissues (including normal and tumoral tissues as well as fetal tissues, 65 samples analyzed) and cell lines. However, human sperm DNA was found unmethylated (Figure 1b).
This assay allows a very rapid screening of the methylation status of a genomic DNA region, and using different sets of primers, a “walk” along the sequence of interest. However, this assay is qualitative rather than quantitative, and in some cases methylation patterns need to be further analyzed by southern blot experiments or by a direct determination of the methylation status of individual CpGs by bisulfite modification of the genomic DNA .
Validation of the bisulfite method
The sodium bisulfite modification method followed by the sequencing of PCR products was used for the determination of the CpG methylation pattern. Sodium bisulfite converts unmethylated cytosines to uraciles while the methylated cytosines remain unmodified. In the resultant modified DNA, uraciles are replicated as thymines during PCR amplification .
After modification, DNA was amplified by a two-step PCR method. The PCR products (position -1,643 to -1,358) were digested by specific restriction endonucleases to determine the global methylation status of the samples. Completeness of the modification was monitored by digestion with Dde I that cleaves only unconverted DNA. PCR products obtained from methylated molecules exhibit a new Eco RI site at position 138, while unmethylated molecules exhibit a new Hph I site at position 165.
The sensitivity of PCR amplification after bisulfite modification was monitored by mixing different proportions of unmethylated DNA from spermatozoa and methylated DNA from HBL 100. For each assay, an aliquot of the PCR product was incubated with Dde I (unmodified DNA); Eco RI (methylated DNA) or Hph I (unmethylated DNA). The results indicate that the amount of PCR product cleaved by enzymatic digestion is directly related to the ratio of methylated/unmethylated DNA used in the bisulfite modification assay (Figure 2).
Analysis by bisulfite sequencing of BRCA1 CpG island
DNAs from somatic tissues and gametes were modified using this method and PCR products were cloned and sequenced. Within the region analyzed, -1,643 to -1,358, the 22 CpG sites analyzed were unmethylated in DNA from human oocytes and spermatozoa (Figure 3). In contrast, these CpGs were methylated in all somatic tissues and cell lines, including the somatic cells of the corona radiata surrounding the oocytes (Figure 3). The absence of DNA methylation within the CpG island in human gametes did not extend to the body of the BRCA1 gene, since control experiments indicated that two regions of the exon 11 are methylated both in somatic tissues and gametes (data not shown), suggesting that the methylation of the CpG island might play a regulatory role in BRCA1 expression.
Bisulfite modification of genomic DNA combined with PCR amplification of the region of interest is an economical method and does not require specialized equipment. The global DNA methylation pattern of a given region can be very easily determined by enzymatic digestion of PCR products. In addition, more precise mapping of methylation patterns can be performed by cloning and sequencing PCR products.
1. Narod SA, Foulkes WD (2004) Nat Rev Cancer 4: 665–676
2. Magdinier F et al. (1998) Oncogene 17: 3169–3176
3. Magdinier F et al. (2000) FASEB J 14: 1585–1594
4. Martin V et al. (1995) Gene 157: 261–264
This article was originally published in Biochemica 3/2006, pages 13-15. ©Springer Medizin Verlag 2006
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