Abstract
In addition to methylated cytosines (5-mCs), hydroxymethylcytosines (5-hmCs) are present in CpG dinucleotide-enriched regions and some transcription regulator binding sites. Unlike methylation, hydroxymethylation does not result in silencing of gene expression, and the most commonly used methods to study methylation, such as techniques based on restriction enzymatic digestion and/or bisulfite modification, are unable to distinguish between them. Genomic imprinting is a process of gene regulation where only one member of an allelic pair is expressed depending on the parental origin. Chromosome 11p15.5 has an imprinting control region (ICR2) that includes a differentially methylated region (KvDMR1) that guarantees parent-specific gene expression. The objective of the present study was to determine the presence of 5-hmC at the KvDMR1 in human placentas. We analyzed 16 third-trimester normal human placentas (chorionic villi). We compared two different methods based on real-time PCR after enzymatic digestion. The first method distinguished methylation from hydroxymethylation, while the other method did not. Unlike other methylation studies, subtle variations of methylation in ICRs could represent a drastic deregulation of the expression of imprinted genes, leading to important phenotypic consequences, and the presence of hydroxymethylation could interfere with the results of many studies. We observed agreement between the results of both methods, indicating the absence of hydroxymethylation at the KvDMR1 in third-trimester placentas. To the best of our knowledge, this is the first study describing the investigation of hydroxymethylation in human placenta using a genomic imprinting model.
Highlights
Genomic imprinting is an epigenetic process involved in the control of gene expression in a parent-of-originspecific manner [1]
The differentially methylated regions (DMRs), where the parental specific methylation takes place, are located within regulatory regions known as imprinting control regions (ICRs)
The 5-hmC is formed from the oxidation of 5-mC by enzymes of the ten-eleven translocation family and, unlike methylation, it does not result in silencing of gene expression [3]. 5-hmCs represent approximately 5% of all cytosines in embryonic stem cells [4], and 20% of all CpGs present in cerebellar Purkinje cells in mammals [5]
Summary
Genomic imprinting is an epigenetic process involved in the control of gene expression in a parent-of-originspecific manner [1]. This functionally haploid state of imprinted genes is essential for normal development of both fetus and placenta. The monoallelic expression of imprinted genes is due to epigenetic mechanisms such as methylation, which consists of the covalent addition of methyl groups by DNA methyltransferases, preferentially at CpG dinucleotides [2]. Several recent studies have shown that, in addition to methylated cytosines (5-mCs), hydroxymethylcytosines (5-hmCs) are present in DMRs and some transcription regulator binding sites [3,4,5]. The 5-hmC is formed from the oxidation of 5-mC by enzymes of the ten-eleven translocation family and, unlike methylation, it does not result in silencing of gene expression [3]. 5-hmCs represent approximately 5% of all cytosines in embryonic stem cells [4], and 20% of all CpGs present in cerebellar Purkinje cells in mammals [5]
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