Placental hydroxymethylation vsmethylation at the imprinting control region 2 on chromosome 11p15.5

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.

Insulin-like growth factor axis in pregnancies affected by fetal growth disorders

“…Another modification of cytosine is called 5-hydroxymethylcytosine (5-HMC) which is also known to play a role in activating or silencing genes [ 50 ]. Liu et al have recently shown significant differences in 5-HMC concentrations in different tissues of the human body with the highest concentration (0.4–0.6 %) found in the brain, liver, kidney and colorectal tissues while lowest concentration (0.02 %) found in the placenta [ 51 ].…”

DNA hydroxymethylation rate in the AChE and HoxC4 promoter associated with human sperm quality

“…The sperm DNA from each individual sample was treated with the use of 5hmC and 5mC analysis Kit (NEB #E3317S), as previously published studies [Davis et al, ; Magalhaes et al, ]. Briefly, genomic DNA was treated with T4 β‐glucosyltransferase (T4‐BGT) at 37°C for 18 hr, adding a glucose moiety to 5hmC which is sequence‐independent; therefore, all 5hmC would be glycosylated, unmodified or 5mC‐containing DNA will not be affected.…”

Hemimethylation of CpG dyads is characteristic of secondary DMRs associated with imprinted loci and correlates with 5-hydroxymethylcytosine at paternally methylated sequences

“…We tested this hypothesis by assessing the relative levels of 5mC and 5hmC at CpGs located in Msp I sites within both primary and secondary DMRs associated with imprinted genes. To conduct this analysis, we glucosylated genomic DNA, digested glucosylated and unglucosylated samples with Msp I, Hpa II or no enzyme, amplified the resulting products using qPCR and calculated percent 5hmC based on the method previously described by Magalhães et al [34]. We conducted our analyses across four developmental stages, and the data shown in Fig.…”

“…qPCR was performed in triplicate for each of the three independent biological samples. Amount of 5mC and 5hmC was calculated according to Magalhães et al [34]. 5hmC levels from each locus were calculated and pairwise combinations of 5hmC levels were ranked and assessed for statistically significant differences between loci using a Mann–Whitney U test (http://vassarstats.net/utest.html).…”