Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue

Abstract

BackgroundGenes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR) which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG) densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes.ResultsRemarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression). Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ-line and the somatic DMRs.ConclusionsOur validated pyrosequencing methylation assays can be widely used as a tool to investigate DNA methylation levels of imprinted genes in clinical samples. This first comprehensive analysis of normal methylation levels in adult somatic tissues at human imprinted regions confirm that, despite intra-individual variability and tissue specific expression, imprinted genes faithfully maintain their DNA methylation in healthy adult tissue. DNA methylation levels of a selection of imprinted genes are, therefore, a valuable indicator for epigenetic stability.

Modeling DNA methylation by analyzing the individual configurations of single molecules

“…[2][3][4][5][6][7][8] Such an approach provides important information on the general relationships between methylation and expression, but it does not look at the specific molecules conformation in each cell. As a consequence, it cannot accurately describe the complex epigenetic structure within a cell population.…”

Erythrocyte folate concentrations, CpG methylation at genomically imprinted domains, and birth weight in a multiethnic newborn cohort

“…Another study limitation is that we examined only nine imprint regulatory sequences, a small fraction of the 65 imprinted genes that are currently known. 74 Moreover, epigenetic changes resulting from variable maternal folate concentrations in early pregnancy are likely not restricted to imprinted domains. Nonetheless, while these data clearly require replication in larger studies, they support the contention that at least for birth weight, excessively high folate concentrations may have no added benefit, and that epigenetic plasticity, measurable at some genomically imprinted DMRs, may mediate these relationships.…”

The DLK1-MEG3 locus in malignant cells of proposed primordial germ cell origins.

“…Interestingly, while the expression of DLK1 is significantly lower in NTera2 cells than in testes, the DLK1/MEG3 ratio is significantly higher in NTera2 cells, suggesting that methylation patterns within this locus may promote the malignant growth of NTera2. This expression ratio agrees with a recent report which found somatic methylation at the DLK1-MEG3 IG-DMR in human testes tissues [124]. What's more, immunostaining of NTera2 cells revealed a strong presence of DLK1 in the nuclei of these cells ( Figure 28).…”

“…Here, I found that the IGF2-H19 ICR is hypomethylated in NTera2 by COBRA as well as by a more sensitive sequencing method ( Figure 24A and 24B, respectively). In contrast, MNCs exhibit higher methylation at this locus, in agreement with a recent report on imprinting in human tissues [124]. Though the exact mechanism of imprinting at the DLK1-MEG3 locus has yet to be determined, the association of methylation at the IG-DMR and MEG3 DMR is similar to that of the IGF2-H19 locus [99].…”

“…I show that, in comparison to MNCs and published data for human testes, this DMR is hypomethylated in mixed TGCTs [124]. What's more, analysis of the IGF2-H19…”