Trisomy 21 alters DNA methylation in parent-of-origin-dependent and independent manners

The supernumerary chromosome 21 in Down syndrome differentially affects the methylation statuses at CpG dinucleotide sites and creates genome-wide transcriptional dysregulation of parental alleles, ultimately causing diverse pathologies. At present, it is unknown whether those effects are dependent or independent of the parental origin of the nondis-joined chromosome 21. Linkage analysis is a standard method for the determination of the parental origin of this aneuploidy, although it is inadequate in cases with deficiency of samples from the progenitors. Here, we assessed the reliability of the epigenetic 5(m)CpG imprints resulting in the maternally (oocyte)-derived allele methylation at a differentially methylated region (DMR) of the candidate imprinted WRB gene for asserting the parental origin of chromosome 21. We developed a methylation-sensitive restriction enzyme-specific PCR assay, based on the WRB DMR, across single nucleotide polymorphisms (SNPs) to examine the methylation statuses in the parental alleles. In genomic DNA from blood cells of either disomic or trisomic subjects, the maternal alleles were consistently methylated, while the paternal alleles were unmethylated. However, the supernumerary chromosome 21 did alter the methylation patterns at the RUNX1 (chromosome 21) and TMEM131 (chromosome 2) CpG sites in a parent-of-origin-independent manner. To evaluate the 5(m)CpG imprints, we conducted a computational comparative epigenomic analysis of transcriptome RNA sequencing (RNA-Seq) and histone modification expression patterns. We found allele fractions consistent with the transcriptional biallelic expression of WRB and ten neighboring genes, despite the similarities in the confluence of both a 17-histone modification activation backbone module and a 5-histone modification repressive module between the WRB DMR and the DMRs of six imprinted genes. We concluded that the maternally inherited 5(m)CpG imprints at the WRB DMR are uncoupled from the parental allele expression of WRB and ten neighboring genes in several tissues and that trisomy 21 alters DNA methylation in parent-of-origin-dependent and -independent manners

Common combinatorial histone modification expression patterns around the <i>WRB</i> CGI-2 DMRs.

<p>Graphical representation of the confluence of activating and repressive epigenetics histone modification marks for the known imprinted <i>MEST</i> gene (<b>A</b>) and the candidate imprinted <i>WRB</i> gene (<b>B</b>). Shown are the 17-histone modification activation backbone module and the 5-histone modification repressive module found in human CD4+ T cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref054" target="_blank">54</a>]. Highlighted in light blue is the DMR in each gene. Composite of screenshots of the dataset viewed at the UCSC Genome Browser hg18 (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>).</p

5^mCpG statuses at the <i>WRB</i> CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line.

<p>A consistent unmethylated pattern of CpG sites at the <i>WRB</i> CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole (<b>A</b>) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line (<b>B</b>). Electropherograms of the amplimers (see details of the assay in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.g002" target="_blank">Fig 2</a>) generated from either undigested DNA or <i>Hha</i>I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.</p

The maternal (oocyte)-derived allele methylation at the <i>WRB</i> CGI-2 distinguishes the parental origin of chromosome 21 nondisjunction events in Down syndrome probands.

<p>The proportion of <i>Hha</i>I-resistant 5^mCpG sites at the <i>WRB</i> CGI-2 DMR in trisomic samples with a maternal origin (MT21) of the nondisjoined chromosome 21 is consistently and statistically higher than in trisomic samples with a paternally (PT21) derived extra copy of chromosome 21. In genomic DNA from an androgenetic hydatidiform mole (ACHM), the <i>WRB</i> CGI-2 is unmethylated, whereas it is hypermethylated in hESCs. In control disomic samples (N21) the <i>locus</i> is partially methylated, consistent with a hemimethylated state. All differences were statistically significant.</p

The methylation statuses at the CpG islands of the <i>WRB</i> gene in methylome public datasets.

<p>Chromosomal, physical map positions and sequence features of the reference <i>WRB</i> gene <i>locus</i> showing the methylation profiles across the region, with an emphasis on the five annotated CpG islands (CGI-1 to CGI-5). The features depicted are from custom and public tracks for (from top to bottom) the exon organization of the principal (ENST00000333781.8) and alternative (ENST00000380708.4) splice <i>WRB</i> isoforms (variants 1 and 2 in dark blue) (UCSC Genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref036" target="_blank">36</a>]), the species-conserved principal transcript (ENST00000333781 in pink) (APPRIS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref075" target="_blank">75</a>]), the Ensembl Regulatory Build CTCF and POL2 activity and predicted promoters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref076" target="_blank">76</a>], CpG islands, CpG sites, and regulation and methylome studies indicated in the Materials and Methods section. The CGI-2 is located in the differentially methylated region (DMR) reported by Court and collaborators [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref024" target="_blank">24</a>] and Docherty and collaborators [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref025" target="_blank">25</a>] (depicted in red in the custom track named Court/Docherty DMR), from which <i>WRB</i> was classified originally as a novel candidate, maternally imprinted gene (i.e., paternal-origin allele expressed). The custom tracks containing the sperm and oocyte DNA methylation signals correspond to the supplementary data reported by Okae <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154108#pone.0154108.ref046" target="_blank">46</a>]. Screenshot generated using the UCSC Genome Browser hg19 (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu</a>).</p

The maternally derived, imprinted 5^mCpG marks at the <i>WRB</i> CGI-2 DMR do not dictate a paternal monoallelic expression in a human embryonic stem cell line.

<p>Allele-specific transcriptional profiling of the <i>WRB</i> 3´-UTR rs1060180 SNP in the informative hESC HUES 1 sample (DNA; upper panel) reveals a pattern consistent with biallelic expression (cDNA; lower panel). In contrast, for the known paternally imprinted <i>H19</i> and <i>ATP10A</i> genes, the expression profiles for the informative rs2839702 and rs2076743 SNPs, respectively, are monoallelic.</p

Experimental validation of the 5^mCpG statuses at the <i>WRB</i> CGIs.

<p>(<b>A</b>) A consistent hemimethylated pattern at the <i>WRB</i> CGI-2 revealed in a representative control disomic DNA sample (blood) using the Hha<i>I</i> methylation-sensitive restriction enzyme-based PCR triplex assay developed in this study. Electropherograms of the amplimers generated from either undigested genomic DNA (upper panel) or DNA digested with <i>Hha</i>I (lower panel) genotyped via quantitative fluorescent PCR. The positive amplimer refers to a <i>locus</i> in the <i>ESCO2</i> gene with constitutively hypomethylated CpG dinucleotides at the target restriction enzyme sites (100% susceptible to <i>Hha</i>I digestion). The negative amplimer refers to a <i>WRB</i> region that lacks <i>Hha</i>I sites, and is, therefore, refractory to enzymatic digestion. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer. In this representative DNA sample, the ratio of 5^mCpG sites at the <i>WRB</i> CGI-2 was 50.6%. In contrast, the assay revealed a consistent unmethylated pattern of CpG sites at the <i>WRB</i> CGI-1 (<b>B</b>) and <i>WRB</i> CGI-3 (<b>C</b>).</p

The maternally derived, imprinted 5^mCpG marks at the <i>WRB</i> CGI-2 DMR do not dictate a paternal monoallelic expression in blood.

<p>Qualitative SNuPE allele-specific profiling of the <i>WRB</i> 3´-UTR rs1060180, rs13230 and rs60490159 SNPs in a disomic, informative nuclear family (DNA). The assay reveals a pattern consistent with biallelic transcriptional expression (cDNA) in the child, who is heterozygous for all three SNP variants.</p

Parent-of-origin-independent methylation effects of the supernumerary chromosome 21 on both syntenic and non-syntenic genes.

<p>(<b>A</b>) A statistically significant gain of methylation at CpG dinucleotide sites within the syntenic <i>RUNX1</i> gene is observed in trisomic individuals compared with control disomic samples (N21). The gain is independent of the maternal (MT21) or the paternal (PT21) origin of the nondisjunction event. (<b>B</b>) A statistically significant loss of methylation at CpG dinucleotide sites within the non-syntenic <i>TMEM131</i> gene is observed in trisomic individuals compared with control disomic samples (N21). The loss is independent of the maternal (MT21) or the paternal (PT21) origin of the nondisjunction event.</p

The allele expression of <i>WRB</i> and ten neighboring genes is uncoupled from the control of the maternally inherited 5^mCpG imprints at the <i>WRB</i> DMR.

<p>(<b>A</b>) UCSC Genome Browser screenshot of custom tracks for the chromosomal and physical map positions of (top to bottom) the 11 genes that map within the 4-Mb chromosomal region centered at the <i>WRB</i> gene, for which evidence for biallelic transcriptional expression was unveiled in this study, and the relative locations of the 162 SNPs queried in RNA-seq public repositories for the determination of allele expression fractions. (<b>B</b>) Density distribution plot, by primary tissue, of the number of SRA accessions of RNA-seq experiments that yielded evidence consistent with biallelic expression. For each gene column, the intensity of the green color of each cell is proportional to the indicated numbers of informative SRA experiments. For each gene, the number of primary tissues with evidence is represented in the bottom row in blue scale.</p