Combined methylation mapping of 5mC and 5hmC during early embryonic stages in bovine

Abstract

BackgroundIt was recently established that changes in methylation during development are dynamic and involve both methylation and demethylation processes. Yet, which genomic sites are changing and what are the contributions of methylation (5mC) and hydroxymethylation (5hmC) to this epigenetic remodeling is still unknown. When studying early development, options for methylation profiling are limited by the unavailability of sufficient DNA material from these scarce samples and limitations are aggravated in non-model species due to the lack of technological platforms. We therefore sought to obtain a representation of differentially 5mC or 5hmC loci during bovine early embryo stages through the use of three complementary methods, based on selective methyl-sensitive restriction and enrichment by ligation-mediated PCR or on subtractive hybridization. Using these strategies, libraries of putative methylation and hydroxymethylated sites were generated from Day-7 and Day-12 bovine embryos.ResultsOver 1.2 million sequencing reads were analyzed, resulting in 151,501 contigs, of which 69,136 were uniquely positioned on the genome. A total of 101,461 putative methylated sites were identified. The output of the three methods differed in genomic coverage as well as in the nature of the identified sites. The classical MspI/HpaII combination of restriction enzymes targeted CpG islands whereas the other methods covered 5mC and 5hmC sites outside of these regions. Data analysis suggests a transition of these methylation marks between Day-7 and Day-12 embryos in specific classes of repeat-containing elements.ConclusionsOur combined strategy offers a genomic map of the distribution of cytosine methylation/hydroxymethylation during early bovine embryo development. These results support the hypothesis of a regulatory phase of hypomethylation in repeat sequences during early embryogenesis.

Oxidative stress in sperm affects the epigenetic reprogramming in early embryonic development

“…4 a, b, 24 h post-IVF the pPN in control zygotes shows a dramatically reduced 5mC signal compared with the mPN, which still retained DNA methylation. These results are consistent with previous reports, showing that also the paternal genome in bovine embryos undergoes DNA demethylation [ 17 , 42 , 78 ]. Remarkably, zygotes fertilized with sperm treated with H 2 O 2 retained higher levels of DNA methylation, indicating that the active DNA demethylation process in the pPN is compromised (Fig.…”

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“…These areas that are below the resolution of conventional confocal microscopy (~100 nm axial and up to ~500 nm vertical) may present captured 5mC-rich sites that were possibly undergoing active DNA demethylation through oxidation and conversion to 5hmC. Several lines of evidence exist that describe DNA methylation and demethylation as dynamic processes, with hypomethylation occurring during the embryonic blastocyst stage and also lasting through differentiation [41,42]. Previously we had observed that up to the sixth day in early differentiation the mouse genome becomes predominantly hypermethylated, first in its euchromatic compartments followed by heterochromatin [7].…”

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