Abstract
Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.
Highlights
Genomic imprinting describes the parent-of-origin dependent monoallelic expression of ∼100–200 genes in mammals
The imprinting control regions (ICRs) is epigenetically marked by DNA methylation in a parent-of-origin fashion, which correlates with expression and/or silencing of the surrounding imprinted genes
The following rules were used for primer design: (i) primer sequences do not contain CpGs to ensure both methylated and unmethylated alleles are amplified ; (ii) the maximum size of amplified regions is ideally 430 bp to reduce any bias introduced from bisulfite treatment-induced DNA fragmentation; (iii) amplified regions contain a minimum of five CpGs and (iv) primers yield a single PCR product when tested on bisulfite-treated genomic DNA (Supplementary Figure S1A, B; Table S2)
Summary
Genomic imprinting describes the parent-of-origin dependent monoallelic expression of ∼100–200 genes in mammals (reviewed in [1]). The inherited set of maternal and paternal chromosomes are not equivalent and are both required for full-term development [2,3]. This effect maps to specific chromosomal regions [4], which later were discovered to contain imprinted genes [5,6,7,8]. The ICR is epigenetically marked by DNA methylation in a parent-of-origin fashion, which correlates with expression and/or silencing of the surrounding imprinted genes. ICRs acquire parental-allele specific DNA methylation in the germline, which are maintained through-
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