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

Abstract

Background: Genes 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.

Results: Remarkable 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.

Conclusions: Our 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.

Figure 1

Average methylation levels for germ-line differentially methylated regions (DMRs; n = 17 in each tissue). Box plots show median, inter-quartile range and maximum/minimum methylation (Bn = brain; Bt = breast; Co = colon; He = heart; Ki = kidney; Li = liver; Pl = placenta). (b) Average tissue methylation levels of somatic DMRs when analysed by parent-of-origin of the methylation. Each data point represents an individual C-phosphate guanine (CpG). Box plots show median, inter-quartile range and maximum/minimum methylation. (c) Intra-CpG variability of DMR methylation. The standard deviation of all CpGs assayed was calculated for each tissue (n = 8). Box plots show median, inter-quartile range and maximum/minimum methylation. Red = maternal germ-line DMRs; pink = maternal somatic DMRs; dark blue = paternal germ-line DMRs; light blue = paternal somatic DMRs; green = parent-of-origin and/or germ-line somatic status unknown.

Figure 2

Analysis of 23 differentially methylated region (DMR) assays in 50 individual blood samples. (a) Average methylation levels of 50 different individuals. Red = maternal germ-line DMRs; pink = maternal somatic DMRs; dark blue = paternal germ-line DMRs; light blue = paternal somatic DMRs. Box plots show the median, inter-quartile range and maximum/minimum methylation B standard deviation of all C-phosphate guanines (CpGs) assayed for maternal and paternal DMRs. A significant difference was observed between the intra-assay variability reported (matched pair t-test, P = 0.007).

Figure 3

Methylation changes at differentially methylated regions (DMRs) after treatment with 5 Azacytidine. (A) Methylation levels of each loci in Sum159 (control) and after treatment with 5 Azacytidine. #1 = 1 nM treatment, #2 = 3 nM treatment. Each data point represents an individual CpG: (i) GNAS XL; (ii) KvDMR; (iii) MCTS2; (iv) MEST (g); (v) RB1; (vi) ZIM2/PEG3; (vii) ZAC; (viii) DIRAS3 (1); (ix) DIRAS3 (2); (x) ZDBF2; (xi) DIRAS3 (3); (xii) GNAS 1A; (xiii) GRB10 (s); (xiv) MEST (s); (xv) NESP55; (xvi) IGF2 (0); (xvii) IGF2 (2); (xviii) MEG3. (b) Percent reduction of methylation at each DMR analysed. Red = maternal germ-line DMRs; pink = maternal somatic DMRs; dark blue = paternal germ-line DMRs; light blue = paternal somatic DMRs. Error bars represent the range of difference between the two experiments.