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

Abstract

Background: It 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.

Results: Over 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.

Conclusions: Our 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.

Figure 1

Schematic illustration of procedures for the three methodologies used to survey embryo methylome and hydroxymethylome. A) Me-RDA and HMe-RDA methodologies use hybridization and subtraction between Tester DNA cleaved with an insensitive enzyme and Driver DNA cleaved with a sensitive isoschizomer. White circle: unmethylated cytosine; orange circle: methylated cytosine. ¹ The subtractive PCR includes a single-strand DNA digestion step by a mung bean nuclease after 10 cycles of amplification and is followed by 20 cycles of amplification. B) HELP Cocktail methodology. Following TasI ligation mediated-PCR, amplification products are digested with a methyl sensitive enzymatic cocktail. Methylated fragments remain uncut, and are subject to exponential amplification in a final PCR.

Figure 2

Resulting smears of amplifications of blastocyst DNA from the three protocols. A) Resulting smears of amplifications from Me-RDA and HMe-RDA methodologies. One round (R1) of kinetic enrichment results in a HpaII site-enriched smear (H) and a BfaI site-enriched smear (B) with PCR control for HpaII (T-H) and PCR control for BfaI (T-B). L: 100 pb ladder. B) Resulting smears from HELP Cocktail methodology. TasI ligation mediated-PCR (PCR1). Final PCR (PCR2).

Figure 3

Distribution of putative methylated/hydroxymethylated restriction sites among the genomic region types. Genomic values are provided as a neutral reference.

Figure 4

Number of methylated/hydroxymethylated restriction sites in the same genomic region for each protocol.

Figure 5

Overlap of methylated/hydroxymethylated genomic regions.

Figure 6

Inter-stage comparison of methylation/hydroxymethylation status of selected subsets of loci. Aliquots of samples were digested using either mark-sensitive or insensitive enzymes. The differences in amplification rates relates to the methylation (5mC or 5hmC) status. Different letters means statistically significant value P < 0.05.