Abstract
Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.
Highlights
DNA methylation is an important epigenetic modification of the genome in many animals and plants
Methylation-specific fluorescence in situ hybridization (MeFISH) is based on the conventional DNA FISH technology for target detection and the ICON technology for 5mC/5hmC detection
Since we successfully applied this method to five different satellites in humans and mice, we believe that it will be useful for detecting methylation at other satellites, including those in other organisms
Summary
DNA methylation is an important epigenetic modification of the genome in many animals and plants. In mammals, it predominantly occurs at the cytosine base of CpG dinucleotides to produce 5-methylcytosine (5mC). DNA methylation patterns are established and maintained by the members of the DNA methyltransferase family (Dnmt, Dnmt3a and Dnmt3b) and their associated factors, including Dnmt3L [1]. DNA methylation plays crucial roles in the regulation of developmental gene expression, chromatin remodeling, genomic imprinting, X-chromosome inactivation and genome stability [2]. Aberrant DNA methylation is an early and fundamental event in the pathogenesis of many human diseases, including cancer [3]. The mechanism of DNA demethylation has been elusive for decades, recent studies revealed that 5-hydroxymethylcytosine (5hmC) is an important intermediate for replication-dependent and/ or replication-independent demethylation [4,5,6]
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