DNA Methylation Polymorphisms Precede Any Histological Sign of Atherosclerosis in Mice Lacking Apolipoprotein E

The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis. The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis. Atherosclerosis and its complications are a major cause of death and disability in the developed world. The disease is characterized by infiltration of lipid particles in the arterial wall, accompanied by the recruitment of inflammatory and immune cells, migration and proliferation of smooth muscle cells (SMC), 1The abbreviations used are: SMC, smooth muscle cells; mC, 5-methylcytosine; CG, CpG dinucleotide; DMP, DNA methylation polymorphism; HPCE, high performance capillary electrophoresis; MSAP, methylation-sensitive amplified polymorphism; SAM, S-adenosyl methionine; PBMC, peripheral blood mononuclear cell; WDMP, wild type DNA methylation polymorphism; MDMP, mutant DNA methylation polymorphism; UK, United Kingdom; EST, expressed sequence tag; WT, wild type; LDL, low density lipoprotein; VLDL, very low density lipoprotein; HDL, high density lipoprotein; HL, high VLDL + LDL mixture; WL, low VLDL + LDL mixture. and synthesis of extracellular matrix. These processes eventually result in the gradual development of an elevated lipid-rich, fibrocellular lesion (1Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar). In mammals, DNA methyltransferases use S-adenosyl methionine (SAM) as a methyl group donor to methylate the carbon in position 5 of cytosine residues in a CpG dinucleotide (CG) context (2Jeltsch A. Chem. Bio. Chem. 2002; 3: 274-293Crossref PubMed Google Scholar). DNA methylation regulates fundamental biological phenomena such as gene expression, genome stability, mutation rate, genomic imprinting, and X chromosome inactivation (3Chen R.Z. Pettersson U. Beard C. Jackson-Grusby L. Jaenisch R. Nature. 1998; 395: 89-93Crossref PubMed Scopus (795) Google Scholar, 4Li E. Nat. Rev. Genet. 2002; 3: 662-673Crossref PubMed Scopus (1584) Google Scholar, 5Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4739) Google Scholar, 6Hashimshony T. Zhang J. Keshet I. Bustin M. Cedar H. Nat. Genet. 2003; 34: 187-192Crossref PubMed Scopus (289) Google Scholar). Both global and gene-specific alterations in DNA methylation are associated with abnormal phenotypes in disease (7Issa J.-P. J. Nutr. 2002; 132: 2388S-2392SCrossref PubMed Google Scholar, 8Feinberg A.P. Tycko B. Nat. Rev. Cancer. 2004; 4: 143-153Crossref PubMed Scopus (1816) Google Scholar). For example, cancer cells show global genomic hypomethylation and dense hypermethylation of CpG islands, which are normally unmethylated (9Esteller M. Herman J.G. J. Pathol. 2002; 196: 1-7Crossref PubMed Scopus (596) Google Scholar). The identification of cancer type- and stage-specific changes in DNA methylation has justified hopes for novel diagnostic and therapeutic avenues (10Laird P.W. Nat. Rev. Cancer. 2003; 3: 253-266Crossref PubMed Scopus (1283) Google Scholar). Two general observations suggest that alterations in DNA methylation patterns are involved in atherogenesis (11Newman P.E. Med. Hypotheses. 1999; 53: 421-424Crossref PubMed Scopus (52) Google Scholar, 12Dong C. Yoon W. Goldschmidt-Clermont P.J. J. Nutr. 2002; 132: 2406S-2409SCrossref PubMed Google Scholar, 13Hiltunen M.O. Ylä-Herttuala S. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1750-1753Crossref PubMed Scopus (98) Google Scholar). First, global hypomethylation and dense hypermethylation of certain CpG islands are associated with aging, a major risk factor for atherosclerosis (14Issa J.-P. Crit. Rev. Oncol. Hematol. 1999; 32: 31-43Crossref PubMed Scopus (169) Google Scholar). Second, hyperhomocysteinemia and the subsequent decreased production or bioavailability of SAM is associated with an increased risk of cardiovascular disease (15Brattström L. Wilcken D.E.L. Am. J. Clin. Nutr. 2000; 72: 315-323Crossref PubMed Scopus (375) Google Scholar). Accordingly, mice with genetically reduced levels of methylenetetrahydrofolate reductase, a key enzyme in the pathway generating SAM, show hyperhomocysteinemia, DNA hypomethylation, and aortic lipid infiltrations (16Chen Z. Karaplis A.C. Ackerman S.L. Pogribny I.P. Melnyk S. Lussier-Cacan S. Chen M.F. Pai A. John S.W.M. Smith R.S. Bottiglieri T. Bagley P. Selhub J. Rudnicki M.A. James S.J. Rozen R. Hum. Mol. Gen. 2001; 10: 433-443Crossref PubMed Scopus (509) Google Scholar). Furthermore, a global DNA hypomethylation has been observed in vascular lesions and leukocytes of atherosclerosis patients and proliferating SMC in animal models (17Laukkanen M.O. Mannermaa S. Hiltunen M.O. Aittomäki S. Airenne K. Jänne J. Ylä-Herttuala S. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2171-2178Crossref PubMed Scopus (142) Google Scholar, 18Hiltunen M.O. Turunen M.P. Hakkinen T.P. Rutanen J. Hedman M. Makinen K. Turunen A.M. Aalto-Setala K. Ylä-Herttuala S. Vasc. Med. 2002; 7: 5-11Crossref PubMed Scopus (205) Google Scholar, 19Castro R. Rivera I. Struys E.A. Jansen E.E.W. Ravasco P. Camilo M.E. Blom H.J. Jakobs C. Tavares de Almeida I. Clin. Chem. 2003; 49: 1292-1296Crossref PubMed Scopus (350) Google Scholar). One unresolved issue is whether DNA methylation patterns are altered at early stages of atherosclerosis and are associated with susceptibility to the disease. Answers to these questions may have important implications for prevention and therapy of atherosclerosis. We therefore asked whether changes in DNA methylation patterns occur prior to the appearance of any vascular lesions in PBMC and aorta of mice lacking APOE, compared with matched WT animals (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar). Moreover, in an attempt to provide a mechanism for the changes in DNA methylation patterns observed in vivo, we asked whether atherogenic lipoprotein profiles could affect DNA methylation and histone post-translational modifications in the human monocyte-macrophage cell line THP-1 (21Auverx J. Experientia (Basel). 1991; 47: 22-31Crossref PubMed Scopus (666) Google Scholar). The present study is the first analysis of DNA methylation at early stages of atherosclerosis and the first description of genomic DNA sequences undergoing epigenetic changes in a mouse model. The implications of our findings for understanding human atherosclerosis are discussed. Animal Work and Tissue Manipulation—All procedures used in this study were approved by the local ethical committee (Malmö/Lunds Djurförsöksetiska Nämnd, license M89-01). Apoe nullizygous mice (Apoe–/–) of the strain created in the laboratory of N. Maeda (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar) were purchased from M&B (Ry, Denmark) and were at the 10th generation of breeding on to the C57BL/6 genetic background. Four-week- and 6-month-old Apoe–/– or C57BL/6 control mice were sacrificed and dissected following overnight fasting. Dissections were performed according to anatomic maps of the mouse (22Popesko P. Rajtova V. Horak J. A Colour Atlas of Anatomy of Small Laboratory Animals. 2. Wolfe Publishing, London, United Kingdom1992: 105-166Google Scholar). The aortic tissue used in this study included the thoracic portion from the middle of the aortic arch and the abdominal portion to the iliac bifurcation. The initial portion of the ascending aorta was used to assess the presence of fatty lesions as described (23Zaina S. Pettersson L. Ahrén B. Brånén L. Hassan A.B. Lindholm M. Mattsson R. Thyberg J. Nilsson J. J. Biol. Chem. 2002; 277: 4505-4511Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). PBMC were isolated by Ficoll-Paque PLUS gradient according to the instructions from the manufacturer (Amersham Biosciences, Little Chalfont, United Kingdom (UK)). Skeletal muscle tissue was dissected from the pelvic limb. Genomic DNA or total RNA were extracted by using the DNeasy or RNeasy system (Qiagen, Valencia, CA), respectively, according to manufacturer instructions. The levels of plasma cholesterol and triglycerides were measured as described (23Zaina S. Pettersson L. Ahrén B. Brånén L. Hassan A.B. Lindholm M. Mattsson R. Thyberg J. Nilsson J. J. Biol. Chem. 2002; 277: 4505-4511Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Methylation-sensitive Amplified Polymorphism (MSAP) Analysis— DNA methylation profiles were analyzed by the MSAP as reported (24Reina-Lopez G.E. Simpson J. Ruiz-Herrera J. Mol. Gen. Genet. 1997; 253: 703-710Crossref PubMed Scopus (356) Google Scholar) with the following modifications. Restriction Digest of Genomic DNA and Attachment of Adaptors—All enzymes used in the MSAP protocol were provided by New England Biolabs (Hertfordshire, UK). Five hundred nanograms of genomic DNA were digested for 1 h with 20 units of HpaII and EcoRI using NEB buffer 1 in a 30-μl reaction volume. Subsequently, the restriction digest and ligation reactions were carried out simultaneously for an additional 3 h in a final volume of 40 μl. The restriction-ligation mix contained 10 units of HpaII, 10 units of EcoRI, 10 units of T4 DNA ligase, 5 pmol EcoRI adaptor, 50 pmol HpaII adaptor and 1 mm ATP. The enzymes were inactivated for 15 min at 65 °C, and a second digest was performed for 1 h with 5 units of HpaII and EcoRI, again followed by heat inactivation. The adaptor sequences were as follows: EcoRI adaptor, 5′-CTCGTAGACTGCGTACC-3′ and 5′-AATTGGTACGCAGTCTAC-3; HpaII adaptor, 5′-GATCATGAGTCCTGCT-3′ and 5′-CGAGCAGGACTCATGA-3′. Preamplification—The preamplification reaction was performed with primers complementary to the core of the adaptor sequences and the target sequence of EcoRI and HpaII restriction enzymes. The sequences of the EcoRI preselective primer (EcoRI-00) and the HpaII preselective primers (HpaII-00) were 5′-AGACTGCGTACCAATTC-3′ and 5′-TCATGAGTCCTGCTCGG-3′, respectively. Two microliters of the digestion-ligation mix (diluted 1:3) were added to the pre-amplification mix consisting of 1× PCR Buffer, 0.1 mm dNTP, 50 ng of EcoRI-00 primer, 50 ng of HpaII-00 primer, and 1 unit of Taq polymerase (Roche Biochemicals). The PCR conditions were as follows: 72 °C for 2 min, 94 °C for 3 min, followed by 25 cycles as follows: 95 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min. A final extension was performed at 72 °C for 4 min. Selective Amplification—The sequence of the selective primers was identical to the preselective primers but included the addition of a number of nucleotides at the 3′ terminus. The selective nucleotides of the EcoRI-00 primer were as follows: EcoRI-01, AGT; EcoRI-02, ACA; EcoRI-03, AGA; and EcoRI-04, ACC. The HpaII selective primers were: HpaII-01, TCCA; HpaII-02, TAGC; HpaII-03, CGAA; HpaII-03A, CGTT; HpaII-04, AATT; and HpaII-04A, AACC. Selective PCR was conducted using 2 μl of pre-amplification mix (diluted 1:10) in a 10-μl reaction volume containing 1× PCR Buffer, 0.1 mm dNTP, 50 ng of EcoRI-00 primer, 50 ng of 33P-labeled HpaII-00 primer, and 1 unit of Taq polymerase. The HpaII primer was end-labeled by incubating 50 ng of primer with 50 μCi of [γ-33P]dATP, 10 units of polynucleotide kinase and 5 μl of 1× OPA buffer (Amersham Biosciences, Buckinghamshire, UK). The reaction was incubated for 1 h at 37 °C, followed by heat inactivation for 15 min at 65 °C. The PCR cycle employed was a standard amplified fragment length polymorphism touchdown protocol (25Vos P. Hogers R. Bleeker M. Reijans M. van de Lee T. Hornes M. Frijters A. Pot J. Peleman J. Kuiper M. Zabeau M. Nucleic Acids Res. 1995; 23: 4407-4414Crossref PubMed Scopus (10559) Google Scholar). Polyacrylamide Gel Electrophoresis—The PCR samples were mixed 1:1 (v/v) with denaturating buffer (98% formamide, 10 mm EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol) and separated on 6% polyacrylamide sequencing gel (Bio-Rad) for 3 h at 90 watts. Gels were dried and exposed to x-ray film (BioMax, Eastman Kodak Co.) for 1–4 days at –80 °C. For each experiment, two or three independent MSAP reactions, each corresponding to pooled samples from three or four mice, were performed. If the results were reproducible, one sample was used for further analysis. Quantitative analyses of band patterns were conducted on central portions of gels, where resolution was maximal. SssI Methyl-accepting Assay—The assay was performed as previously described (26Schmitt F. Oakeley E.J. Jost J.P. J. Biol. Chem. 1997; 272: 1534-1540Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), with the exception that Nonidet P-40 was replaced with Triton and labeled DNA was blotted on a Nytran SuPerCharge membrane (Schleicher & Schuell, Dassel, Germany). Isolation and Sequencing of MSAP Bands—Bands were excised from acrylamide gels, suspended in 30 μlof0.5× TE buffer, and incubated for 10 min at 65 °C. One microliter of the solution was used in a standard PCR reaction with the appropriate primer combinations. The resulting fragments were cloned in the pCR®II-TOPO® vector (Invitrogen, Paisley, UK), and three independent clones were sequenced. Homology searches were conducted using NIX software (www.hgmp.mrc.ac.uk). The CpG ratio was calculated by dividing the number of observed CG dinucleotides by their expected number, calculated with the formula (number of cytosines)(number of guanines)/(total number of nucleotides). Southern Blotting and Methylation-sensitive PCR Analysis of DMR— Fifteen micrograms of genomic DNA were digested overnight with the appropriate restriction enzyme, blotted, and probed with selected labeled MSAP fragments. Hybridization was conducted by using the ULTRAhyb™ system according to instructions from the manufacturer (Ambion, Huntingdon, UK). Selected fragments for which flanking sequences could be deduced from data base matches were analyzed by methylation-sensitive PCR (27Santourlidis S. Florl A. Ackermann R. Wirtz H.-C. Schulz W.A. The Prostate. 1999; 39: 166-174Crossref PubMed Scopus (155) Google Scholar). Two hundred nanograms of DNA was amplified with the following fragment-specific primers. Fragment sz44 consisted of internal primer, 5′-TAGCAGAGACCTAGAAGAGGG-3′ (forward); and flanking primer, 5′-TTCAAAAGTTGCCTCAAGTCC-3′ (reverse), corresponding to nucleotides 70314–70294 of sequence with accession no. AC103664. Fragment M54 consisted of internal primer, 5′-TGTGTTTTCCTCTCTTAGCCC-3′ (forward); and flanking primer, 5′-TTCTCAGCCATTCGGTATTCC-3′ (reverse), corresponding to nucleotides 86183–86163 of sequence with accession no. AC120437. Fragment M4 consisted of internal primer, 5′-AGTGCCTGTGATCCTTACCTG-3′ (forward); and flanking primer, 5′-AAAGCTCAGAGTAGAAAAGGG-3′ (reverse), corresponding to nucleotides 194754–194774 of sequence with accession no. AC132863. Conditions were as follows: 94 °C for 1 min; followed by 35 cycles at 94, 51, and 72 °C, each for 1 min; and a final extension step at 72 °C for 10 min. Predicted PCR fragments were sz44, 399 bp; M54, 327 bp; and M4, 282 bp. Expression Analysis by Reverse Transcription-PCR—Expression analysis of leukocytic MSAP polymorphic fragments showing homology to EST or genomic sequences (see "Results") was conducted by using fragment-specific nested primers. Gapdh RNA was amplified as an internal control (Clontech, Palo Alto, CA). Primer identity and PCR conditions are available upon request. Southern Blotting Analysis of LINE-1 Elements—A 548-bp probe spanning a conserved 5′ region sequence in mouse LINE-1 elements (28Furano A.V. Prog. Nucleic Acids Res. Mol. Biol. 2000; 64: 255-294Crossref PubMed Google Scholar) was constructed by subjecting mouse tail genomic DNA to PCR with the primers 5′-ATCTTGGTTCGGGACCCGCCGAACTTAGG-3′ (forward) and 5′-GTTTACCTTTCGCCATCTGGTAATCTCTGG-3′ (reverse). Conditions were as follows: 94 °C for 1 min; followed by 35 cycles at 94, 60, and 72 °C, each for 1 min; and a final extension step at 72 °C for 10 min. The probe was sequenced and used in Southern blotting as indicated in the previous paragraph. Cell Culture and Lipoprotein Isolation—Human THP-1 cells were maintained in RPMI 1640 medium supplemented with fetal calf serum (10%), glutamine (4 mm), penicillin (20 IU/ml), and streptomycin (20 μg/ml). Cells were either used as monocytes or differentiated to macrophages by incubation with phorbol myristate acetate (50 ng/ml) for 4 days with change of medium every second day. For experiments, both monocytes and macrophages were incubated for 24 h in serum-free media with lipoprotein additions as indicated under "Results." Lipoproteins were prepared by differential ultracentrifugation of fresh plasma from healthy donors (29Lindholm M. Sjoblom L. Nordborg C. Ostlund-Lindqvist A.M. Eklund A. Ann. Nutr. Metab. 1993; 37: 302-310Crossref PubMed Scopus (8) Google Scholar). Quantification of Total 5-Methylcytosine by High Performance Capillary Electrophoresis (HPCE)—Quantification of the degree of methylation was carried out as previously described (30Fraga M.F. Uriol E. Borja Diego L. Berdasco M. Esteller M. Canal M.J. Rodríguez R. Electrophoresis. 2002; 23: 1677-1681Crossref PubMed Scopus (137) Google Scholar, 31Ballestar E. Paz M.F. Valle L. Wei S. Fraga M.F. Espada J. Cigudosa J.C. Huang T.H. Esteller M. EMBO J. 2003; 22: 6335-6345Crossref PubMed Scopus (284) Google Scholar). Briefly, genomic DNA (3–5 μg) was obtained by standard procedures and DNA hydrolysis was carried out with 1.25 μl (200 units/ml) of nuclease P1 for 16 h at 37 °C. Subsequently, alkaline phosphatase was added and mixtures were incubated for an additional 2 h at 37 °C. Hydrolyzed samples were injected under pressure (0.3 p.s.i.) for 3 s into an uncoated fused-silica capillary in a CE system (P/AC™ MDQ, Beckman-Coulter). Quantification of the relative methylation of each DNA sample was determined as the percentage of 5-methylcytosine (mC) of total cytosines: mC peak area × 100/(C peak area + mC peak area). All samples were analyzed in duplicate, and three analytical measurements were made per replicate. Quantification of Whole Histone H4 Acetylation and Methylation of Lysine 20 by HPCE—Quantification of acetylation and methylation at lysine 20 of histone H4 was done by a modification of the method previously described (32Lindner H. Helliger W. Dirschlmayer A. Talasz H. Wurm M. Sarg B. Jaquemar M. Puschendorf B. J. Chromatogr. 1992; 608: 211-216Crossref PubMed Scopus (39) Google Scholar). Individual histone fractions were obtained from cell nuclei (33Goodwin G.H. Nicolas R.H. Johns E.W. Biochem. J. 1977; 167: 485-488Crossref PubMed Scopus (17) Google Scholar), and further purified by reversed-phase HPLC (34Gurley L.R. Prentice D.A. Valdez J.G. Spall W.D. Anal. Biochem. 1983; 131: 465-477Crossref PubMed Scopus (25) Google Scholar). The non-, mono-, di-, tri-, and tetra-acetylated forms of histones H3 and H4 and trimethylated H4 at lysine 20 were resolved by HPCE, using an uncoated fused-silica capillary (Beckman-Coulter™) (60.2 cm × 75 mm, effective length 50 cm) in a CE system (P/ACE™ MDQ, Beckman-Coulter™). The running buffer was 100 mm phosphate buffer, pH 2.0, containing 0.02% (w/v) HPM-cellulose. Running conditions were 25 °C and operating voltages of 12 kV. On-column absorbance was monitored at 214 nm. Before each run, the capillary system was conditioned by washing with 0.1 m NaOH for 3 min, and with 0.5 m H2SO4 for 2 min, and equilibrated with running buffer for 3 min. Buffers and washing solutions were prepared with Milli-Q water and filtered throughout 0.45-μm filters. Samples were injected under pressure (0.3 p.s.i.) for 3 s. All samples were analyzed in duplicate, and three analytical measurements were made per replicate. Statistics—Comparisons were made by using nonparametric tests in the StatView (Abacus Concept, Berkeley, CA) or Statistica (StatSoft, Tulsa, OK) program for Macintosh. The counts of DNA methylation polymorphic bands were compared in samples paired by selective primer, using the Wilcoxon paired test. In all other cases, tests are specified under "Results." Changes in PBMC and Aortic DNA Methylation Profiles Precede Fibrocellular Vascular Lesions in Apoe–/– Mice—To study DNA methylation patterns during the progression of atherosclerosis, 4-week-old and 6-month-old APOE-null (Apoe–/–) were used. Mice were fed normal rodent diet, to exclude any confounding contribution of dietary factors potentially affecting DNA methylation (35Friso S. Choi S.-W. J. Nutr. 2002; 132: 2382S-2387SCrossref PubMed Google Scholar). At the age of 4 weeks, mutant mice were hypercholesterolemic but lacked any detectable fatty streak or fibrocellular lesions at the ascending aorta or at the aortic arch (Fig. 1, A and B). By contrast, lesions consisting of a fibrocellular intima and a lipid-rich core were detectable in the same portion of the aortic vessel in 6-month-old Apoe–/– mice as previously reported (Fig. 1, C and D) (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar). Plasma total cholesterol was markedly elevated in Apoe–/– mice of both age groups, relative to WT controls (Fig. 1, compare A and C with B and D) (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar). To screen PBMC for DNA methylation polymorphisms (DMPs) between Apoe–/– mice and matched controls, we exploited the MSAP fingerprinting technique. This technique is a modification of amplified fragment length polymorphism, a procedure that is based on random amplification of restriction fragments typically generated by digestion of genomic DNA with the EcoRI and MseI restriction enzymes (25Vos P. Hogers R. Bleeker M. Reijans M. van de Lee T. Hornes M. Frijters A. Pot J. Peleman J. Kuiper M. Zabeau M. Nucleic Acids Res. 1995; 23: 4407-4414Crossref PubMed Scopus (10559) Google Scholar). In MSAP, MseI is replaced by the methylation-sensitive enzyme HpaII, blocked by methylation at either cytosine residue in the recognition site 5′-CCGG-3′ (24Reina-Lopez G.E. Simpson J. Ruiz-Herrera J. Mol. Gen. Genet. 1997; 253: 703-710Crossref PubMed Scopus (356) Google Scholar). Following digestion of genomic DNA, adaptors are attached to restriction sites that have been successfully digested. It follows that the products of the MSAP ligation reaction consist of DNA fragments that are flanked by hypomethylated HpaII and EcoRI sites. Thereafter, two consecutive PCR reactions, a preamplification and a selective amplification, are performed to enrich a subpopulation of the restriction fragments. The primers employed are complementary to the core sequence of adaptors and recognition sites of the restriction enzymes, and the number of nucleotides added to their 3′ terminus determines their selectivity. Typically the number of selective nucleotides is increased in the selective amplification, where one of the two primers is radioactively labeled. This enables the visualization of a subset of restriction fragments by autoradiography following acrylamide gel electrophoresis. PBMC DNA was screened by MSAP, using 23 different combinations of selective primers, corresponding to a total of ∼1,600 bands/genotype group. Parallel MSAP analyses were conducted with MspI, an isoschizomer of HpaII that is only sensitive to methylation at the external cytosine (methylation at CNG trinucleotide) on one or both strands (methylation on one strand only is referred to as hemimethylation). It follows that an HpaII polymorphism between WT and Apoe–/– samples can be attributed to differential CG methylation, if the corresponding MspI profile is monomorphic, i.e. either present or absent in both genotypes (Fig. 2A). In the latter case, the HpaII site is hemimethylated at the external cytosine in both genotypes (lower bands in Fig. 2A) (36McClelland M. Nelson M. Raschke E. Nucleic Acids Res. 1994; 22: 3640-3659Crossref PubMed Scopus (354) Google Scholar). Alternatively, absence of MspI fragments may result from inefficient amplification of the more complex MspI amplicons, relative to HpaII products. Nonetheless, the stability of MspI profiles observed throughout our study argues against this hypothesis. A polymorphism detected with both HpaII and MspI digestion is indicative of CNG methylation or mutation at the targeted Apoe alleles or elsewhere (Fig. 2B). The analysis revealed that polymorphisms were present at both 4 weeks and 6 months of age (Fig. 3). Although DMPs may contain internal hypermethylated HpaII or EcoRI sites, previous analyses revealed that the vast majority of MSAP products represent HpaII-EcoRI fragments that lack any internal HpaII or EcoRI site (37Lauria M. Rupe M. Guo M. Kranz E. Pirona R. Viotti A. Lund G. Plant Cell. 2004; 16: 510-522Crossref PubMed Scopus (88) Google Scholar). Accordingly, in the present study, only 5% of the sequenced MSAP fragments (see below) contained an internal HpaII site and none contained any EcoRI site. This implies that hypermethylation of internal fragment sites affects only marginally, if at all, the assessment of genome methylation status by MSAP band scoring. Two hundred and eight of the 206 polymorphisms observed at 4 weeks and 6 months (counting polymorphisms that are common to both ages only once) represented changes in CG methylation, e.g. genuine DMPs (Fig. 3, A and B), whereas only one band represented a difference in CNG methylation or a mutation (Fig. 3C). The total number of DMPs in 4-week-old PBMC was 48, or 3.2% of total bands. A similar analysis conducted on 6-month-old mice revealed that the total number of DMPs was increased by 3.8-fold to 185, or 11% of total bands, compared with the younger age group (p < 0.002). DMPs that were present in both age groups represented a substantial part of all DMPs at 4 weeks (27 of 48, or 56%), but were a minority at 6 months (15%). An identical MSAP analysis was conducted on aortic DNA, using 14 selective primer combinations, producing a total of ∼1,000 bands. Aortic DMPs showed a markedly different distribution, in comparison with PBMC DNA. The total number of DMPs showed little or no increase with age, being 76 and 83 (or 7.6% and 8.3% of total bands) at 4 weeks and 6 months, respectively. Furthermore, approximately one third of DMPs were conserved in both age groups. The comparison of PBMC and aortic MSAP analyses revealed that few DMPs were common to the PBMC and the aorta (2 of 137 total aortic DMPs at 4 weeks and 6 months, counting polymorphisms that are common to both ages only once). Further MSAP analysis was conducted on pericardial fat and liver of 6-month-old mice by using six primer combinations. In these tissues, the frequency of DMPs was severalfold lower than in matched PBMC and aorta (i.e. 0.5–1% of total bands; p < 0.03 in all comparisons). This indicates that the bulk of DMPs observed in Apoe–/– mice from the age of 4 weeks on represented genotype-related and tissue-specific, rather than reflecting inter- or intra-individual epigenetic variations that are unrelated to the Apoe genotype (see, for example, Ref. 38Yatabe Y. Tavaré S. Shibata D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10839-10844Crossref PubMed Scopus (295) Google Scholar). Hypo- and Hypermethylation in Apoe–/– Mice—A DMP that is characterized by a band specifically present in Apoe–/– mutant (MDMP) or WT (WDMP) mice, implies hypo- or hypermethylation at the flanking HpaII site, respectively, in Apoe–/– mice compared with WT. Previous comparisons of parallel results obtained with MSAP and direct quantitation of mC by HPCE, established that the total number