Chromosome hydroxymethylation patterns in human zygotes and cleavage-stage embryos

Abstract

We report the sequential changes in 5-hydroxymethylcytosine (5hmC) patterns in the genome of human preimplantation embryos during DNA methylation reprogramming. We have studied chromosome hydroxymethylation and methylation patterns in triploid zygotes and blastomeres of cleavage-stage embryos. Using indirect immunofluorescence, we have analyzed the localization of 5hmC and its co-distribution with 5-methylcytosine (5mC) on the QFH-banded metaphase chromosomes. In zygotes, 5hmC accumulates in both parental chromosome sets, but hydroxymethylation is more intensive in the poorly methylated paternal set. In the maternal set, chromosomes are highly methylated, but contain little 5hmC. Hydroxymethylation is highly region specific in both parental chromosome sets: hydroxymethylated loci correspond to R-bands, but not G-bands, and have well-defined borders, which coincide with the R/G-band boundaries. The centromeric regions and heterochromatin at 1q12, 9q12, 16q11.2, and Yq12 contain little 5mC and no 5hmC. We hypothesize that 5hmC may mark structural/functional genome 'units' corresponding to chromosome bands in the newly formed zygotic genome. In addition, we suggest that the hydroxymethylation of R-bands in zygotes can be treated as a new characteristic distinguishing them from G-bands. At cleavages, chromosomes with asymmetrical hydroxymethylation of sister chromatids appear. They decrease in number during cleavages, whereas totally non-hydroxymethylated chromosomes become numerous. Taken together, our findings suggest that, in the zygotic genome, 5hmC is distributed selectively and its pattern is determined by both parental origin of chromosomes and type of chromosome bands -R, G, or C. At cleavages, chromosome hydroxymethylation pattern is dynamically changed due to passive and non-selective overall loss of 5hmC, which coincides with that of 5mC.

Inter-Cell and Inter-Chromosome Variability of 5-Hydroxymethylcytosine Patterns in Noncultured Human Embryonic and Extraembryonic Cells

“…Hydroxymethylated zygotic chromosomes passively lose 5hmC during a replication cycle, which results in chromosome hemihydroxymethylation. With cleavages, hemihydroxymethylated chromosomes are gradually substituted by nonhydroxymethylated chromosomes due to further replication-dependent 5hmC loss, which coincides with that of 5mC Inoue and Zhang, 2011;Pendina et al, 2011;Efimova et al, 2015b]. Random segregation of hydroxymethylated and nonhydroxymethylated chromatids results in different chromosome hydroxymethylation patterns in each blastomere [Efimova et al, 2015b].…”

“…The immunodetection of 5hmC and 5mC was performed according to the protocol used previously [Efimova et al, 2015b]. In brief, the preparations were denatured in 2 M HCl for 20-30 min, washed thoroughly in ice-cold PBS and distilled water, and incubated with blocking solution (1% BSA, 0.1% Tween 20 in PBS) for 30-40 min at 37 ° C. The mixture of antibodies against 5hmC (rabbit polyclonal, Active Motif, 39769, Carlsbad, CA, USA) and 5mC (mouse monoclonal, Millipore, clone 33D3, MABE146, USA) diluted in blocking solution (1: 250) was applied to the slides for 90 min at room temperature.…”

“…However, the passive loss of 5hmC in blastomeres is linear due to the absence of new global hydroxymethylation events over the entire period of preimplantation development [Inoue and Zhang, 2011;Efimova et al, 2015b]. In contrast, chromosome hydroxymethylation patterns in postimplantation embryonic and extraembryonic cells point towards nonlinear fluctuations of 5hmC levels.…”

Genomic distribution of 5‐formylcytosine and 5‐carboxylcytosine in human preimplantation embryos

“…These regions were mainly located subtelomerically and had distinct borders that corresponded to T‐band boundaries. Such highly discrete localization contrasted substantially with that of methylated and hydroxymethylated DNA, which was distributed along the whole euchromatic chromosome arms (Efimova, Pendina, Tikhonov, et al, ; Pendina et al, ). Intriguingly, around two‐thirds (69%) of the identified nucleolar organizing regions (NORs), which were located in the short arms of acrocentric autosomes, were also highly enriched in 5fC and, especially, in 5caC.…”

“…Intriguingly, around two‐thirds (69%) of the identified nucleolar organizing regions (NORs), which were located in the short arms of acrocentric autosomes, were also highly enriched in 5fC and, especially, in 5caC. This particular feature distinguished 5fC and 5caC patterns from those of 5mC and 5hmC, because the latter were not pronounced in the ribosomal DNA (rDNA) regions (Efimova, Pendina, Tikhonov, et al, ; Pendina et al, ). In the maternal chromosome sets, the distribution of 5fC and 5caC was similar to that in the paternal ones, but their intensity was considerably lower (Figure a).…”