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

Silvio Zaina,Esteban Ballestar,Manel Esteller,Ana Villar-Garea,Linda Andersson,Marie Lindholm,Gertrud Lund,Mario F Fraga,Massimiliano Lauria

doi:10.1074/jbc.m403618200

Abstract

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.

Highlights

The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis
To screen PBMC for DNA methylation polymorphisms (DMPs) between ApoeϪ/Ϫ mice and matched controls, we exploited the Methylation-sensitive Amplified Polymorphism (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 [25]
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

Summary

EXPERIMENTAL PROCEDURES

Animal Work and Tissue Manipulation—All procedures used in this study were approved by the local ethical committee (Malmo/Lunds Djurforsoksetiska Namnd, license M89-01). 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-␮l reaction volume. Selective PCR was conducted using 2 ␮l of pre-amplification mix (diluted 1:10) in a 10-␮l reaction volume containing 1ϫ 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. 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. Southern Blotting Analysis of LINE-1 Elements—A 548-bp probe spanning a conserved 5Ј region sequence in mouse LINE-1 elements [28] was constructed by subjecting mouse tail genomic DNA to PCR with the primers 5Ј-ATCTTGGTTCGGGACCCGCCGAACTTAGG-3Ј (forward) and 5Ј-GTTTACCTTTCGCCATCTGGTAATCTCTGG-3Ј (reverse).

RESULTS

Type of sequence bp

DISCUSSION