Methylation pattern of polymorphically imprinted nc886 is not conserved across mammalia.

BACKGROUND: In humans, the nc886 locus is a polymorphically imprinted metastable epiallele. Periconceptional conditions have an effect on the methylation status of nc886, and further, this methylation status is associated with health outcomes in later life, in line with the Developmental Origins of Health and Disease (DOHaD) hypothesis. Animal models would offer opportunities to study the associations between periconceptional conditions, nc886 methylation status and metabolic phenotypes further. Thus, we set out to investigate the methylation pattern of the nc886 locus in non-human mammals. DATA: We obtained DNA methylation data from the data repository GEO for mammals, whose nc886 gene included all three major parts of nc886 and had sequency similarity of over 80% with the human nc886. Our final sample set consisted of DNA methylation data from humans, chimpanzees, bonobos, gorillas, orangutangs, baboons, macaques, vervets, marmosets and guinea pigs.
RESULTS: In human data sets the methylation pattern of nc886 locus followed the expected bimodal distribution, indicative of polymorphic imprinting. In great apes, we identified a unimodal DNA methylation pattern with 50% methylation level in all individuals and in all subspecies. In Old World monkeys, the between individual variation was greater and methylation on average was close to 60%. In guinea pigs the region around the nc886 homologue was non-methylated. Results obtained from the sequence comparison of the CTCF binding sites flanking the nc886 gene support the results on the DNA methylation data.
CONCLUSIONS: Our results indicate that unlike in humans, nc886 is not a polymorphically imprinted metastable epiallele in non-human primates or in guinea pigs, thus implying that animal models are not applicable for nc886 research. The obtained data suggests that the nc886 region may be classically imprinted in great apes, and potentially also in Old World monkeys, but not in guinea pigs.

Background

In mammalian genomic imprinting, only one parental allele is expressed, while gene expression from the other allele is suppressed in a parent-of-origin-dependent manner. Imprinting has been hypothesized to have evolved in response to a parent-offspring conflict, where the maternal and paternal genome differ in their interests regarding the supply of resources [1]. Paternal genomes favor the opportunistic strategy to enhance the growth of the developing offspring through the expression of growth-enhancing genes, while maternal genomes aim to conserve maternal resources over multiple pregnancies [2]. On the other hand, according to the trophoblast defense theory, suppression of genes that promote placental development or to activate genes that limit this process in oocytes are thought to protect the females from parthenogenetically activated oocytes and consequential malignant trophoblast [3, 4]. The expression of imprinted genes in general has been associated with fetal and placental growth and suggested to a have a role in the development of cardiometabolic diseases in adulthood [2, 4, 5]. Imprinting arose relatively recently at most loci—while only a few imprinted genes in Eutherians are also imprinted in marsupials, to date no imprinting has been reported in the egg-laying monotreme mammals [6]. This supports the conflict-of-interest theory, as only in non-egg laying mammals the growing fetus directly consumes the maternal resources [4]. Genetic imprinting is best described in mice, and while the mouse is an informative proxy for human imprinted gene regulation, less than half of the 100 human imprinted genes have been shown to be similarly imprinted in mice (https://www.geneimprint.com/site/home). Distinct differences in placental evolution, physiology, and reproductive biology of the primate and murine groups may be responsible for these differences.

During gametogenesis and fertilization, the original DNA methylation pattern of imprinted genes is erased, and parent of origin-based methylation pattern is established. While most imprinted genes are located in clusters that are regulated by insulators or long noncoding RNAs [3], some unclustered imprinted genes can be regulated by differential promoter methylation [4]. Parental imprints are maintained after fertilization through these mechanisms despite extensive reprogramming of the mammalian genome [4]. A common feature of imprinted genes is insulators, such as CCCTC binding factor (CTCF) binding sites, which block the enhancers from interacting with gene promoters and/or act as barrier to the spread of transcriptionally repressive condensed chromatin [7].

The locus harbouring non coding 886 (nc886, also known as VTRNA2-1) in chromosome 5q31.1 is a unique example of imprinting, as it is the only known polymorphically imprinted locus across tissues in adult human population, where the polymorphism is not due to genetic variation [8-12]. However, in the placenta polymorphic imprinting is more common [13, 14]. The nc886 differentially methylated region (DMR) is 1.9-kb long, and its boundaries are marked by two CTCF binding sites [10, 15]. This DMR has been shown to present maternal imprinting in ~75% of individuals in several populations [10, 12, 16]. This means that while in all individuals the paternal allele is unmethylated, in approximately 75% of individuals the maternal allele is methylated (individuals present a 50% methylation level at nc886 locus) and in the remaining 25% of individuals the maternal allele is unmethylated (individuals present a 0% methylation level at the nc886 locus).

The nc886 gene codes for a 102nt long, non-coding RNA, which is then cleaved into two short RNAs (hsa-miR-886-3p/nc886-3p [23 nt] and hsa-miR-886-5p/nc886-5p [24–25 nt]) [17-19]. There is no consensus on whether the effects of nc886 expression is mediated by the 102 nt long hairpin structure or the nc886-3p and -5p molecules, as the short molecules have been indicated to function as miRNAs, while the hairpin loop has been shown to inhibit protein kinase R (PKR) [17, 20]. Expression of nc886 RNAs is strongly associated with the methylation status of the nc886 locus. Individuals with non-methylated nc886 present approximately two-fold levels of nc886 RNAs in blood, as compared to individuals with monoallelic methylation [16], thus implying allele-specific expression. However, allele-specific expression has not been experimentally shown, as the 102 nt transcript does not harbour SNPs [10].

The periconceptional environment has been suggested to affect DNA methylation patterns in maternal alleles [21], including the nc886 epiallele [10, 15, 16]. Season of conception, maternal age and socioeconomic status have been linked to changes in the proportion of offspring with unmethylated maternal allele [10, 15, 16]. On the other hand, lower levels of nc886 methylation have been linked to cleft palate [22], and a non-methylated nc886 epiallele has been associated with an elevated childhood BMI [23]. The methylation status of this epiallele has also been associated with allergies [24], asthma [25], infections [26], and inflammation [27]. We and others have also shown that both the nc886 methylation status and RNA expression are associated with indicators of glucose metabolism [16, 28].

These results indicate that nc886 could mediate the association between periconceptional conditions and later metabolic health, in line with the Developmental Origins of Health and Disease (DOHaD) hypothesis (aka the Barker hypothesis) [29]. More detailed analysis on periconceptional conditions and nc886 methylation status and investigations between nc886 and metabolic phenotypes, with less cofounding factors, would be needed to confirm this hypothesis. As carcinogenesis [9] and pluripotency induction [16] affect the DNA methylation pattern in nc886 locus, in vitro work has its limitations. Animal models could be a feasible option for this research. Unfortunately, rodents do not harbor the nc886 gene, limiting the use of traditional model organisms [10]. Thus, this study was set up to investigate 1) which animals have nc886 gene, 2) whether this gene is surrounded by similar CTFC elements as the human homolog and 3) whether the methylation status of the nc886 region suggest polymorphic imprinting in non-human mammals.

Materials and methods

The presence of nc886 gene was investigated in ensemble, in 65 amniota vertebrates Mercator-Pecan collection and 24 primates EPO-extended collection [30]. To select species for further investigation, we required the nc886 gene have 80% sequence similarity with the human homolog and to present the sequences for nc886-3p and nc886-5p RNAs, as well as the loop structure, previously shown to mediate the binding of PKR [17, 31] (S1 and S2 Figs). The existence and sequence similarity of the centromeric (chr5:135415115–135415544) and telomeric CTCF (chr5:135418124–135418523) binding site flanking nc886 gene were also investigated in species shown to harbor intact nc886 gene. If homologous CTCF-binding sites were not discovered, CTCFBSDB 2.0 [32] was utilized to predict possible non-homologous sites. Interactions of the nc886 flanking CTCF-sites were also investigated using K562 CTCF ChIA-PET Interactions data and Hi-C data in genome browser [33] and Hi-C data in 3D Genome Browser from HUVEC and K562 cell lines [34].

For species harboring the nc886 gene, we investigated the Gene Expression Omnibus (GEO) repository [35] for available DNA methylation data, with both general and binomial name of the species. DNA methylation data was available in apes from chimpanzees (Pan troglodytes, n = 83; GSE136296 [36] and n = 5; GSE41782 [37]), bonobos (Pan paniscus, n = 6; GSE41782 [37]), gorillas (Troglodytes gorilla, n = 6; GSE41782 [37]) and orangutangs (Pongo spp., n = 6; GSE41782 [37]). In Old World monkeys, data was obtained from baboons (Papio spp. n = 28; GSE103287 [38]), rhesus macaques (Macaca mulatta, n = 10; GSE103287 [38]), vervets (Chlorocebus aethiops, n = 10; GSE103287 [38]) and in New World monkeys, from marmosets (Callithrix jacchus, n = 6; GSE103287 [38]). From primates, only data from blood or femur was utilized. In addition to primates, we obtained DNA methylation data from guinea pig hippocampus (Cavia porcellus, n = 36; GSE109765 [39]). As reference, we utilized data from human (Homo sapiens) blood (n = 1658; GSE105018 [40]), femur (n = 48; GSE64490 [41]) and hippocampus (n = 33; GSE72778 [42]).

Methylation profiling data obtained by high throughput sequencing (guinea pigs, GSE109765) was processed as follows. Quality of the paired-end reads in all the samples was assessed using FastQC [43] and MultiQC [44]. Paired-end fastq files were trimmed using Trimmomatic-0.39 with a sliding window of size 4 set to remove bases with phred score lower than 20 [45]. The trimmed samples were analyzed using Bismark-0.23.0 tools [46]. The reads were aligned to the guinea pig genome (cavPor3). Duplicate alignments, which can arise for example by PCR amplification, were removed. Methylation information was extracted from the alignment result files using Bismark’s methylation extractor. DNA methylation values for CpGs inside the gene were first inspected and then a wider region (±2000nt) around the gene was investigated.

Primate DNA methylation data from GSE41782, GSE105018, GSE64490, GSE72778 (profiled with Illumina 450K) and GSE136296 (profiled with Illumina EPIC) were available as processed data and was used as such. Primate DNA methylation data from GSE103271, GSE103280, GSE103286, which are subseries of GSE103332 (profiled with Illumina EPIC), were available as raw data, and were normalized by using minfi quantile normalization for each species separately.

From primate data, the 14 CpGs in the nc886 DMR previously reported to show bimodal methylation pattern in humans were retrieved [10, 16]. In all the primate species, for which methylation data was available, the sequence on the binding site of the Illumina probes was investigated. Only data from sites with the CG-sequence intact in the species in question were further utilized. (S1 File). Similar process was repeated for 50 probes in paternally expressed 10 (PEG10) previously shown to be imprinted [47]. PEG10 was used as evolutionally conserved reference for a classically imprinted gene [6].

Results and discussion

nc886 gene in non-human mammals

Human nc886 has been suggested to be an evolutionally young gene, producing a 102 nt long RNA, which is then ineffectively cleaved to two miRNA-like RNAs [17]. In line with previous reports [10], nc886 gene, with intact short RNA coding sequences and the hairpin loop, can be found in primates, in guinea pig (Cavia porcellus), Eurasian red squirrel (Sciurus vulgaris), and Alpine marmot (Marmota marmota), with two of the latter having insertions in the centromeric end of the gene (Figs 1 and 2, S1 Fig, S1 Table). In humans, and all the other species where nc886 was found, nc886 is located between TGFB1 and SMAD5.

Fig 1

Schematic presentation of the similarity of the nc886 gene and flanking CTCF binding sites.

Guinea pigs lack the whole centromeric flanking CTCF binding site, whilst the marmosets have a region with sequence similarity, but lack the binding sequence of the CTCF. Mouse genome does not contain either the nc886 gene or the CTCF binding sites flanking it. For detailed sequence comparisons, see S1 and S2 Figs.

Fig 2

Schematic presentation of the nc886 locus.

In red, the nc886 gene (chr5:135416180–135416287), in green the centromeric CTCF binding site (chr5:135415115–135415544) and in blue the telomeric CTFC binding site (chr5:135418124–135418523). The binding motif of the CTCF sites are presented in yellow and the 14 CpGs with bimodal methylation pattern in human cohorts are presented according to their genomic location (GRCh37/hg19).

Upon further inspection of primate nc886 gene, almost 100% sequence similarity was identified in apes (hominoidea). The sequence similarity was high (over 97%) between humans and the investigated Old World monkeys (Cercopithecidae), while less (over 90%) similarity can be seen between humans and New World monkeys (Ceboidea) and even less (84–89%) between humans and tarsiers (Tarsiidae) or lemurs (Lemuroidea). Sequences coding for the short nc886 RNAs are identical within Old World anthropoids (Catarrhini). Differences between human and New World monkey and prosimian nc886 sequences can be found both in the short nc886 RNA and the hairpin coding regions, but the most significant differences can be found in the centromeric end of the gene (Fig 1 and S2 Fig).

CTCF binding site sequence in mammals with nc886 gene

In humans, the nc886 locus is flanked by two CTCF binding sites (Fig 2) [10]. The telomeric CTCF binding site can be found in most vertebrates, with the binding motif being identical in all species showed to have the nc886 gene (S1 Table). The telomeric CTCF binding site was shown to interact with a CTCF binding site (chr5:135222814–135223707) locating near the IL9 gene in the CTCF ChIA-PET data, the interaction is also supported by the Hi-C data from cell lines (S3 Fig). This prediction is in line with our previous finding indicating that genetic polymorphisms near IL9 gene are associated with the expression of nc886 RNAs [16]. This CTCF binding site locating near IL9 can be found in all primates (S1 Table). Together these results suggest that the evolutionally conserved telomeric CTCF binding site of nc886 interacts with CTCF binding sites near IL9 gene, possibly forming a topologically associating domain (TAD), or an interaction within one (sub-TAD), and bringing the suggested enhancer area near the nc886 gene [48]. On the contrary, the centromeric CTCF binding site is present only in primates and even in primates, the binding sequence cannot be identified in marmosets. As CTCF binding sites can act as barriers to the spread of transcriptionally repressive condensed chromatin [7], presence, or absence, of the CTCF binding site may be associated with the methylation status of nc886 locus in different species. For the centromeric CTCF binding site no interactions were detected according to CTCF ChIA-PET data. There are also changes in the CTFC binding motif in gorillas (position 9), in all Old World monkeys (position 4) and also in New World monkeys (position 14) (S1 Table). According to the CTCFBSDB 2.0, the guinea pig genome does not harbor any non-homologous predicted CTCF binding sites in the centromeric side of the nc886 gene.

Guinea pigs present a non-methylated nc886 locus

One of the aims of this study was to investigate whether the information gathered from the nc886 gene and DMR from cell culture and population studies could be supplemented with research on animal models. In line with a previous report [10] we identified this gene only in primates, guinea pigs and few members of the squirrel family, of which guinea pig was the most promising candidate as a model organism. In data from Constantinof et al. [39], in guinea pig hippocampi (n = 36) the whole nc886/vtRNA2-1 gene was non-methylated (S4 Fig). The surrounding nc886 region (Scaffold DS562872.1: 24,622,179–24,622,280) +/- 2000 nt was mostly unmethylated, with only 2% of the reads in the region being methylated. It should be noted that the number of reads in the region in the data utilized was low (max number of reads = 17, average number of reads = 7). nc886 methylation pattern in human hippocampi presented the expected bimodal distribution, and thus the discovered methylation pattern in the guinea pig hippocampi was most likely not due to the selection of tissue (S4 Fig). This identified lack of methylation in the nc886 locus is compatible with the absence of the telomeric CTCF binding site, as CTCF binding sites can delineate the boundaries of an imprinted region [49]. These results thus suggest that guinea pigs are not suitable model organisms for the investigation of establishment of nc886 methylation status.

Imprinted nc886 region in great apes

The blood of chimpanzees, gorillas, bonobos and orangutans presented beta-values close to 0.5 with a unimodal distribution in the nc886 region (Fig 3, S2 Table). This methylation pattern closely resembles the methylation pattern in the known maternally imprinted gene PEG10 (S5 Fig). The methylation levels are also very similar to those presented in humans with monoallelic methylation (Fig 3). In these data sets, that include more than 110 apes, we did not identify any individuals with methylation level close to 0 in the nc886 locus, whereas in humans 25% of the population present a methylation level close to 0 at this locus [10, 16]. If the prevalence on non-methylated individuals in apes was similar to humans, already 11 individuals would present at least one non-methylated individual with 95% probability. Of individual species, we had the largest dataset for chimpanzees (n = 83 in GSE136296 and n = 5 in GSE41782). Again, assuming the same proportion of non-methylated individuals as in humans (25%), probability of not identifying any non-methylated chimpanzees in a population of 88 individuals is extremely low, 1.01*10−11. These results imply that the nc886 locus is not polymorphically imprinted in apes.

Fig 3

Blood DNA methylation beta values in nc886 region in great apes (Hominidae).

In each graph, one dot represents one individual. In humans a bimodal methylation pattern can be detected, in line with the expected population distribution of individuals with monoallelic methylation (75% of the population, methylation level ~0.5) and non-methylated individuals (25% of the population, methylation level close to 0) [10,16]. All of the other species present a unimodal methylation pattern, with all individuals having methylation beta-values near 0.5 across the nc886 locus. Number of individuals is 1658 for humans (GSE105018), 6 for bonobos (GSE41782), 83 and 5 for chimpanzees (GSE136296 and GSE41782, respectively), 6 for gorillas (GSE41782) and 6 for orangutans (GSE41782). For schematic representation of the CpG sites in the nc886 locus, see Fig 2.

Within species, median of standard deviation of the probe methylation of nc886 is around 0.04, which is comparable to that seen in PEG10 in apes (median of SD 0.03) and in human blood (0.02), indicating good data quality (S2 Table). In all apes the methylation levels of nc886 region resembled those seen in PEG10, which is an evolutionally conserved maternally imprinted gene [6]. It is thus reasonable to suggest that nc886 could be classically imprinted in other great apes, excluding humans.

nc886 region methylation patterns in Old World monkeys

The patterns of nc886 region methylation are very similar in all of the Old World monkeys. The median methylation level is close to 0.60, higher as compared to apes. The interindividual variation is larger, especially in baboons, than in apes, with the median SD within a probe being 0.11. The SD within a probe is also higher in probes locating in PEG10 in baboons, where the median of probe SD is 0.05 (S2 Table). As the between individual variation in methylation levels of a known evolutionally conserved imprinted gene is also higher, this suggests a technical bias in the data, potentially due to the use of Illumina Infinium 450K and EPIC methylation assays, that are designed for humans. All methylation data available for Old World monkeys was from femur, but as the methylation pattern of nc886 has been reported to be constant across tissues in humans [12] and in the human reference data set form femur samples both nc886 and PEG10 present similar methylation patterns as in blood (Fig 4 and S6 Fig), this phenomenon most likely is not caused by the tissue of origin. Regardless of the precise methylation levels of the Old World monkeys, in the 48 individuals we did not identify any presenting a non-methylated methylation pattern in nc886 region, probability of which is 1.0*10−6, when assuming similar distribution as in humans. This implies that similar to non-human great apes, the nc886 locus is not polymorphically imprinted in Old World monkeys.

Fig 4

Femur DNA methylation beta values in the nc886 region in humans and in monkeys.

In humans a bimodal methylation pattern can be detected, similar to blood and in line with the expected population distribution of individuals with monoallelic methylation (75% of the population, methylation level ~0.5) and non-methylated individuals (25% of the population, methylation level close to 0) [10, 16]. In Old World monkeys the between individual variation is bigger than in apes. No individuals presenting a non-methylated nc886 region are detected. In marmosets only 3 probes were considered to provide reliable methylation values and none of them present methylation levels near 0.5. Number of individuals is 48 for humans (GSE64490), 28 for baboons (GSE103287), 10 for macaques (GSE103287), 10 for vervets (GSE103287) and 6 for marmosets (GSE103287). For schematic representation of the CpG sites in the nc886 locus, see Fig 2.

nc886 region methylation patterns in New World monkeys

Data from the six marmosets is not conclusive, as we found that only 3 of the probes in nc886 region bound to areas with no great sequence differences. The methylation levels of two of these probes were around 0.8 and one 0.32, showing no indications of imprinting, while the median of methylation beta values in the probes locating in the PEG10 is 0.46 in marmosets (Fig 4 and S6 Fig, S2 Table). The lack of imprinting of any kind in marmosets is further supported by the finding that they lack the centromeric CTCF binding motif, which is thought to have an important role in insulating the DMR [10]. To draw conclusions on the methylation status of nc886 in New World monkeys in general, methylation data from more species would be needed.

Limitations of the study

Our study is purely descriptive in nature. In guinea pigs, the shallowness of the sequencing data limits the ability to make conclusions of the methylation pattern. In non-human primates, utilizing methylation arrays that have been designed for humans raises questions on data quality, especially in marmosets. Concerns over data quality are however mitigated by the observed methylation pattern in well-established imprinted gene, PEG10, as well as the consistency of observed results across different species and data sets. In addition, while a methylation level of 0.50 implies allele specific methylation, we were not able to confirm this due to lack of suitable data.

Conclusions

We describe here an analysis on the methylation status of nc886 region in non-human mammals. A genetic locus, with more than 80% similarity to human nc886 gene can be found in primates, guinea pigs and some members of the squirrel family. We obtained DNA methylation data for 8 different non-human primate species and for guinea pigs, and in none of these species could we observe methylation pattern indicative of similar polymorphic imprinting, as could be observed, and has been reported [10, 12, 16], in humans. The observed methylation pattern in apes and in Old World monkeys implies that the nc886 region might be classically imprinted, although these findings have to be interpreted with caution, as apart from chimpanzees the sample number were low, and in Old World monkeys the variation between individuals was notable. In guinea pigs, the most feasible potential model organism of those harboring the nc886 locus, the data indicated that the locus is completely unmethylated. It is noteworthy that only primates, whose genome also contained the centromeric CTCF binding sequence flanking the nc886 gene, had methylation levels indicative of genetic imprinting.

In conclusion, we were unable to identify an animal model suited to study the establishment of the methylation pattern of the polymorphically imprinted metastable epiallele nc886. Further studies on how this kind of unusual metastable developed, and how it links to the periconceptional conditions and later life health traits, are thus restricted to in vitro and population studies.

Declarations

Ethics approval

Datasets used in this study were retrieved from Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo/) repository. For human data sets, informed consent or “Consent for Autopsy” were given by all participants. For animal studies all protocols were approved by the local ethical committees and/or the samples were collected during standard veterinary checks or routine necropsies. Details can be found from the original publications [36-42].

Sequence alignment of the nc886 gene in the 65 amniota vertebrates Mercator-Pecan.

To state that a species presents the nc886 we required 80% sequence similarity with the human nc886 and presence of both nc886-5p and nc886-3p short RNAs and the hairpin loop present in the 102nt long nc886 RNA. Species above the dashed line were considered to harbour the full nc886 gene. Note: Sequence alignment figures are presented in the direction of the gene. Species with no identified alignment in this region were excluded from the figure (Sauropsids, opossum (Monodelphis domestica) and platypus (Ornithorhynchus anatinus)).

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Sequence alignment of the nc886 gene in the 24 primates in EPO-extended collection.

Sequence similarity to human nc886 gene decreases as evolutionary distance increases and greatest diverge is seen in the centromeric end of the gene. Note: Sequence alignment figures are presented in the direction of the gene.

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Predicted interaction between nc886 telomeric CTCF binding site and CTCF binding site located near IL9.

A) ChIA-PET data and in HI-C data in Genome browser cell lines and in 3D genome browser. B) HUVEC and C) K562 cell lines. The suggested sub-TAD has been indicated with a black square and interactions in HI-C data have been circumscribed. The telomeric CTCF binding site is located at chr5:135418124–135418523 and the CTCF binding site near IL9 at chr5:135223050–135223420.

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Methylation of nc886 region in hippocampi of A) guinea pigs (GSE109765) and B) humans (GSE64509).

In (A) each circle presents a CpG site with measured methylation level and each line one individual guinea pig (n = 36). The number of reads per site is low (on average 7) but none of the samples provide data that would indicate anything but non-methylated DNA methylation status around the guinea pig nc886 gene. In human hippocampi (B) the methylation pattern of nc886 presents the expected binomial methylation pattern, which is in line with reported frequency of 25% non-methylated individuals and 75% individuals with monoallelic methylation in a population.

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Methylation pattern of Paternally expressed 10 (PEG10) in blood of great apes.

Only probes locating in sites with no clear sequence differences as compared to the human PEG10 sequence are shown.

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Methylation pattern of Paternally expressed 10 (PEG10) in femur bone of humans and Old and New World monkeys.

Only probes locating in sites with no clear sequence differences as compared to the human PEG10 sequence are shown.

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Sequence similarities of nc886 gene and its centromeric and telomeric CTCF binding sites, and the CTCF binding site located near Il9 gene in primates, marmot, squirrel, guinea pig and in mouse investigated from NCBI BLAST and ensemble.

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Methylation medians and standard deviations of the PEG10 and nc886 regions and individual probes in the nc886 region in primates.

Medians and standard deviations in human nc886 are not comparable to other species, as it is known to demonstrate bimodal methylation pattern in humans.

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Sequence alignment of the nc886 region between human, bonobo, chimpanzee, gorilla, orangutang, gibbon, baboon, macaque, vervet and marmoset.

The sequence comparison covers the region containing the 14 CpG sites presenting bimodal methylation pattern in humans (chr5:135415593–135416666). These CpG sites are highlighted in yellow.

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