Distinct Patterns of Gene-Specific Methylation in Mammalian Placentas: Implications for Placental Evolution and Function
Article Outline
- Abstract
- 1. Introduction
- 2. Methods
- 3. Results
- 4. Discussion
- Acknowledgements
- Appendix. Supplementary data
- References
- Copyright
Abstract
The placenta has arisen relatively recently and is among the most rapidly evolving tissues in mammals. Several different placental barrier and structure types appear to have independently evolved common functional features. Specific patterns of gene expression that determine placental development in humans are predicted to be accompanied by specific profiles of epigenetic modification. However, the stratification of epigenetic modifications into those involved in conserved aspects of placental function, versus those involved in divergent placental features, has yet to begin. As a first step towards this goal, we have investigated the methylation status of a small number of gene-specific methylation events recently identified in human placenta, in a panel of placental tissue from baboon, marmoset, cow, cat, guinea pig and mouse. These represent disparate placental barrier types and structures. In this study we hypothesized that specific epigenetic markings may be associated with placental barrier type or function, independent of phylogeny. However, in contrast to our predictions, the majority of gene-specific methylation appears to track with phylogeny, independent of placental barrier type or other structural features. This suggests that despite the likelihood of epigenetic modification playing a role in the functioning and evolution of different placental subtypes, there is no evidence for an involvement of the gene-specific methylation profiles we have identified, in specifying these differences. Further studies, examining larger numbers of epigenetic modifications across phylogeny, are required to define the role of specific epigenetic modifications in the evolution of distinct placental structures.
Keywords: DNA methylation, Epigenetics, Wnt signalling, DNMT1, Vitamin D 24-hydroxylase
1. Introduction
In addition to regulating nutrient exchange between maternal to fetal circulations, the placenta represents the first line of defense in protecting the developing fetus from adverse environmental exposures and the maternal immune system. Although all placentas share a basic common function in modulating exchange at the fetomaternal interface, it is clear that the human placenta is unique in structure and function [1].
Placentas can be classified in several ways, including by the number of cell layers separating maternal and fetal bloodstreams [2]. There is a maximum of six potential cellular layers, three maternal and three fetal. Epitheliochorial placentas contain all six layers and are found in the horse, pig and ruminant species. However, the ruminant placenta is strictly synepitheliochorial in which trophoblast binucleate cells migrate away from the fetal trophoblast layer and fuse with maternal uterine luminal epithelial cells to form a fetomaternal syncytium. Endotheliochorial placentas, with one maternal layer and three fetal layers, are present in carnivores and some insectivores, lower primates, and megachiropteran bats. Hemochorial placentas, found in higher primates, rodents, microchiropteran bats and the armadillo, show a more extensive trophoblast invasion during development that remodels maternal arteries, thereby maximizing blood flow to the intervillous space [3].
Each eutherian order/sub order has a distinctive placental shape and type of fetomaternal attachment that have presumably evolved to meet specific fetal needs. Therefore, despite the value of animal models in examining fundamental functions of the placentation process, they may be of limited value in fully dissecting molecular processes contributing to specific human pathologies such as pre eclampsia (PE), intra uterine growth restriction (IUGR) and babies small for gestational age (SGA) [4]. The mechanisms underlying the distinct differences within eutherian placentas have remained elusive but clearly include a unique gene expression profile in different species (reviewed in [5]). Limited evidence also supports a role of distinct epigenetic modification in placental functioning and morphology across evolution.
It has been known for many years that the placenta displays some unique epigenetic features compared to somatic cells. This includes the lowest 5-methylcytosine content (∼3.1%) of all human tissues except germ cells [6], [7], [8], comparable to that in human cancers [9], [10], [11], and a unique set of imprinted genes (expressed in a parent-of-origin manner) regulated by DNA methylation [12]. Many of these genes are involved in regulation of growth, with disruption of imprinting previously associated with IUGR [13], SGA [14], PE [15], [16], [17], and the use of assisted reproductive technologies [18], [19], [20], [21], [22].
More recently, non-imprinting-associated placenta-specific epigenetic modification has been described (eg. [23], [24]). This includes several instances of tumour suppressor (TS) gene silencing by histone modification and/or DNA methylation [25], [26], [27], [28]. TS genes are generally unmethylated in healthy human somatic tissue but are often methylated and silenced in human cancers. Despite the fact that general disruption of DNA methylation by specific drug treatment disrupts placental development and inhibits invasiveness in trophoblast cell lines [29], [30], [31], for the most part, the function of placenta-specific epigenetic modification remains to be elucidated.
In this study we investigated the association of a small number of gene-specific methylation events, previously described in humans, with distinct features of eutherian placentas such as barrier type and shape. Genes of interest are involved in disparate functions including Wnt pathway signaling, vitamin D homeostasis, and maintenance of DNA methylation. These pathways play pivotal roles in modulation of trophoblast invasiveness, differentiation, and immune functioning and differing regulation of these genes via epigenetic divergences may be associated with variations in previously described placental morphology and trophoblast function.
2. Methods
2.1. Tissue samples
Human tissues were harvested for research purposes with appropriate human ethics clearances from the Royal Women's Hospital (03/51), Mercy Hospital for Women (R07/15), and Monash Medical Centre (07084C). Full term baboon placental tissue was provided by Dr Neroli Sunderland (Royal Prince Alfred Hospital, Sydney Australia, approval 2009/028A). Full term marmoset placental tissue was obtained from Monash Animal Services (Melbourne, Australia). Full term guinea pig placenta was obtained with ethical approval from the University of Adelaide Animal Ethics Committee (M-73-2003). Day 153 cow cotyledon tissue was collected from the Placenta fetalis with approval from the University of Adelaide Animal Ethics Committee (S-094-2005A). Day 18 gestation mouse placental tissue was provided by Drs Patrick Western and Craig Smith (Murdoch Childrens Research Institute, Australia), and 4 week gestation cat placental tissue was obtained from Dr Chris Thurgood following routine desexing (Royal Society for the Protection and Care of Animals, Burwood, Victoria, Australia). These tissues were provided in line with the principle of Reduction, NHMRC Code of Practice for the Care and use of animals for scientific purposes, 7th Edition, 2004. For human placental tissue sampling, a core of full thickness tissue was isolated and bisected into two layers from maternal and fetal sides. All placental tissues were processed by maceration with a scalpel blade immediately prior to DNA isolation.
2.2. Genomic DNA isolation
Tissue samples were incubated at 50 °C overnight with shaking in DNA extraction buffer [100 mM NaCl, 10 mM TrisHCl pH8, 25 mM EDTA, 0.5% SDS] with 200 μg/mL Proteinase K. DNA was isolated by two rounds of phenol:chloroform extraction, followed by RNAse A treatment, precipitation in absolute ethanol containing 10% sodium acetate (3M, pH 5.2), and resuspended in 50 μL TE Buffer [10 mM TrisHCl pH7.5, 1 mM EDTA]. DNA was quantitated and quality assessed by mass spectroscopy on a Nanodrop (ThermoFisher Scientific, Waltham MA, USA), and stored at −20 °C.
2.3. Methylation analysis using bisulphite DNA sequencing
DNA samples were processed using the MethylEasy™ bisulphite modification kit (Human Genetic Signatures, North Ryde, NSW, Australia) kit according to the manufacturer's instructions. Sodium bisulphite selectively converts unmethylated cytosine (C) residues to uracil nucleotides. This covalent modification is stably maintained during subsequent PCR amplification where the uracil is converted to the complementary thymidine nucleotide included in the amplification mix. PCR amplification was performed on converted genomic DNA using primers directed to bisulphite modified DNA (sequences listed in Supplementary Table 1). Assays for each region of interest were designed following bioinformatic analysis to identify equivalent regions to that previously assayed in humans (Fig. 1). In the event that assay design parameters did not allow equivalent regions to be assessed, regions of close proximity were examined. Although bisulphite sequencing is relatively time consuming in comparison to other methods, and measures a small number of alleles within any tissue specimen, it remains the ‘gold standard’ approach for the parallel characterization of both DNA methylation level and distribution at specific loci. This was an important consideration in the current study where both aspects of DNA methylation were being assessed.
Fig. 1
Location of methylation assays used in the current study. Alignment of methylation assays was performed using the BLAT function of the UCSC genome browser (human genome build GRCh37). Chromosomal location is listed along with the relative positions and level of sequence homology between the different methylation assays. The 5′ end of genes of interest is shown along with the transcription start site (arrow). Red dashes denote individual CpG sites within each assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Amplification conditions were 95 °C for 5 min, 56 °C for 1 min 30 s and 72 °C for 1 min 30 s × 40 cycles, 72 °C for 7 min. Resulting amplicons were cloned into TOPO TA Cloning kit (Invitrogen, Carlsbad, CA, USA) for automated fluorescent sequencing as previously described [32]. Data were analyzed using BiQ Analyser software [33] and clones showing less than 80% conversion or 90% homology to the reference sequence were not included in subsequent analyses.
3. Results
We used bisulphite sequencing to profile the methylation status of a small number of gene promoters in placental tissue from several eutherians, representing widely divergent placental morphologies, barrier types and phylogenetic distances. Using this approach, we found that patterns of methylation for the four genes analyzed could be broadly classed into 5 groups as detailed in Table 1.
Table 1. Classification of methylation profile according to mean absolute methylation level and distribution pattern.
| Class | Absolute methylation levela |
| HIGH | >70% |
| MED | 30–70% |
| LOW | <30% |
| Evidence for monoallelic methylation | Criteriab |
| STRONG (MONO) | Three or more instances of coincident fully methylated plus fully unmethylated alleles |
| WEAK (mono?) | At least one instance of coincident fully methylated plus fully unmethylated alleles |
| NONE | No examples of coincident fully methylated plus fully unmethylated alleles |
aAbsolute methylation cutoffs for each class are arbitrarily defined. |
bFully methylated in this instance was defined was defined as >90% methylation, while fully unmethylated was <10% methylated CpG sites within single alleles as determined by bisulphite sequencing. |
3.1. Comparative methylation of Wnt pathway regulators
The Wnt signaling pathway plays a pivotal role in many cellular functions, including cell division, proliferation, differentiation and motility [34]. Active Wnt signaling has been implicated in the survival, differentiation, and invasion of human trophoblasts with nuclear β–catenin accumulation apparent in extravillous trophoblast cells [35].
Previous studies from our laboratory have shown that the major promoter of the Adenomatous Polyposis Coli (APC) gene (negative regulator of Wnt signaling) is monoallelically methylated specifically in human (hemomonochorial villous) placenta and purified trophoblasts, with no methylation apparent in mouse (hemotrichorial labyrinthine) placental tissues [27]. We examined the methylation status of this promoter in baboon (hemomonochorial villous), marmoset (hemomonochorial villous), cow (synepitheliochorial cotyledonary), cat (endotheliochorial zonary) and guinea pig (hemomonochorial labyrinthine) placental tissue. Whereas strong evidence of APC monoallelic methylation was found in baboon placental tissue, manifesting as intermediate (MED) methylation levels (MED-MONO class), almost complete methylation (HIGH) was apparent in marmoset placental tissue, with no evidence of a monoallelic pattern (HIGH class; Fig. 6B). In contrast, much lower levels of methylation (LOW) were detected in cow, cat and guinea pig placental tissue, with weak evidence of a minor population of highly methylated alleles in each of these species (LOW-mono? class). Essentially no methylation of this region was detected in mouse placentas (LOW class; Figs. 1and 6B).
We also examined methylation of the SFRP2 gene (representing another class of negative regulators of Wnt signaling), previously measured in human placental tissue [28]. When the corresponding region was investigated in other eutherian placentas, highly variable methylation levels and profiles were found in different species. This included intermediate mean methylation levels in baboon, marmoset and guinea pig placentas with strong evidence of monoallelic methylation (MED-MONO class) in marmoset with little or no evidence for monoallelic methylation in individual baboon and guinea pig placental tissue (MED-mono? class; Figs. 2 and 6B). Lower levels of methylation were seen in the cow, with some evidence for a minor population of highly methylated alleles (LOW-mono?), whereas this region in the cat and mouse placentas were both essentially unmethylated (LOW; Figs. 2 and 6B).
Fig. 2
Methylation of the APC gene in 7 eutherian placentas. Following bisulphite conversion of placental genomic DNA, regions of interest were amplified with primer sequences specific to converted DNA sequence. Amplified PCR products were then cloned into a DNA vector before transformation into E. coli. Individual colonies were then randomly selected for DNA isolation and sequencing. Biological replicate samples from independent placentas (number listed in parentheses) were tested for each species. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. Grey horizontal bars denote CpG island regions associated with the gene of interest and transcription start site (raised arrows). Black bars show locations of associated exonic regions. Red dashes denote individual CpG sites within each assay which correspond to circles below. Circled methylation data are presented for a single placental sample (as an example) for which 8–12 DNA sequences from individual alleles (horizontal rows) were generated. Due to space constraints, only 8 alleles from each placental sample are shown. Shaded circles denote methylated CpG sites within specific DNA clones (open circles). Mean methylation (0–100%) for each CpG site in biological replicates (number in brackets) is shown as shaded bars below each site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.2. Comparative methylation of the vitamin D catabolic 24-hydroxylase gene (CYP24A1)
Very little is known about the epigenetic regulation of enzymes modulating vitamin D homeostasis at the fetomaternal interface. However, we recently identified placenta-specific methylation of the CYP24A1 gene in human placental tissue and purified first trimester cytotrophoblasts [23]. Given the likely importance of vitamin D in proper placental development and functioning, fetal development, and in the modulation of maternal immune response to the developing fetus, we speculated that epigenetic regulation of this gene may be conserved across all eutherians.
An examination of the methylation level in marmoset placental tissue revealed a pattern of intermediate methylation with no evidence for monoallelic methylation (MED), in contrast to that seen in human full term placenta and purified trophoblasts (MED-MONO) [23]. Surprisingly, similar levels were also observed in guinea pig placenta with weak evidence for a minor population of highly methylated alleles in some placentas (MED-mono?). Lower levels of methylation were observed in baboon, similar to that in the mouse (LOW; Fig. 3). Essentially no methylation was detected in the corresponding region of the CYP24A1 gene in cat and cow placental tissue (LOW; Figs. 3 and 6B).
Fig. 3
Methylation of the SFRP2 gene in 7 eutherian placentas. Bisulphite sequencing was carried out as described in Fig. 2. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. For details see Fig. 2.
3.3. Primate-specific DNMT1 promoter methylation in eutherian placental tissue
The family of DNA methyltransferases DNMT1, -3A, -3B and -3L are responsible for the establishment and maintenance of DNA methylation in mammals (reviewed in [36]). We have recently demonstrated high prevalence monoallelic methylation of the DNMT1 gene in human and baboon placental tissue [55]. In this study we have confirmed high prevalence monoallelic methylation in human and marmoset placental tissue (MED-MONO; Fig. 4) and demonstrated a similar pattern in baboon full term placentas (also MED-MONO). Much lower levels of methylation were seen in cow, cat, guinea pig and mouse placental samples (LOW; Fig. 4) demonstrating a primate-specific pattern of promoter methylation (Fig. 5).
Fig. 4
Methylation of the CYP24A1 gene in 7 eutherian placentas. Bisulphite sequencing was carried out as described in Fig. 2. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. For details see Fig. 2.
Fig. 5
Methylation of the DNMT1 gene in 7 eutherian placentas. Bisulphite sequencing was carried out as described in Fig. 2. Data presented for (A) human, (B) baboon, (C) marmoset, (D) cow, (E) cat, (F) guinea pig, and (G) mouse placental tissue. For details see Fig. 2.
Fig. 6
Placenta specific gene methylation levels according to phylogenetic relationship (A) Phylogenetic relationship of eutherians showing placenta barrier type (He, Hemochorial; Hm, Hemomonochorial; Hd, Hemodichorial; Ht, Hemotrichorial; Ep, Epitheliochorial; En, Endotheliochorial), placenta shape (C; Cotyledonary; Z, Zonary; D, Diffuse; Dd, Discoid; Bd, Bidiscoid), and type of interdigitation (T, Trabecular; Lb, Labyrinthine; F, Folded; L, Lamellar). Species examined in this study are highlighted in red. (B) Summary of methylation level for each of the four genes examined in this study for seven different species. Phylogenetic tree adapted from {Wildman, 2006 #1672}. Note that the classification of manatee placental barrier type has recently been modified from He to En {Carter, 2008 #1673}. Classification of methylation profile is based on parameters listed in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
4. Discussion
Despite diverse structural differentiation and some functional parameters in placentas from different placentas, all share several common features. Interspecific comparison of the placental epigenome therefore offers an unparalleled opportunity to identify molecular mechanisms contributing to both conserved and divergent placental functions.
Previous limited epigenetic analysis has identified a general hypomethylation of extraembryonic tissue in humans (lower genomic 5-methylcytosine content) relative to somatic tissues due to a more pronounced re-methylation of the embryonic lineage very early in development [7], [8], [37], [38], [39]. This phenomenon is also evident in placental tissue of baboon, marmoset, guinea pig [55], mouse [39], [40], [41], rabbit [42], sheep [43], and cow [44], [45], and where examined, is correlated with lower levels of methylation at repetitive DNA sequences.
Accumulated data have demonstrated a link between genomic imprinting (an epigenetic phenomenon), the evolution of viviparity and the acquisition of a placenta in metatherian and eutherian mammals, but it is also clear that the spectrum of placental imprinting varies between species. For example, genes imprinted in both embryonic and extraembryonic tissues show extensive conservation between mouse and human and are generally regulated by DNA methylation [12]. In contrast, many genes show placenta-specific imprinting in mice, but not humans. Such genes are invariably regulated at the level of histone modification rather than DNA methylation [46].
Placenta-specific gene expression has been documented over many years. This has revealed potential conserved gene expression signatures associated with placentation, but also interspecific differences in genes such as transcription factors that likely lead to differential regulation of a wide range of downstream genes and cellular processes (reviewed in [5]). Despite intensive efforts to catalogue genes specifically expressed in the placenta, no systematic examination of genes showing placenta-specific down-regulation (or silencing) has been reported. Such silencing is usually mediated by epigenetic modification.
Formation of any tissue, including the placenta, requires a coordinated series of epigenetic modifications that precisely regulate gene expression at key developmental time points (reviewed in [47]). DNA methylation is a widely studied epigenetic regulator of gene expression, and disruption of placental methylation has been unequivocally linked to aberrant placental function. Specifically, 5-deoxy azacitidine (5-Aza; general DNA methylation inhibitor) administered to pregnant rats, disrupts trophoblast proliferation and pregnancy development [30], [31], and 5-Aza treatment of choriocarcinoma-derived cell lines in vitro inhibits trophoblast migration [29]. In addition, gene knockout studies of DNA methyltransferase enzymes Dnmt1 or Dnmt3l are associated with placental defects [48], [49].
Recent large scale-studies have begun to profile locus-specific placental methylation on a genome-wide (or subgenomic) level in humans [50], [51], [52], [53]. Other studies have begun to characterize specific methylation directly in the placenta in a variety of species (eg. [25], [28], [54]). However, the level of conservation of gene-specific methylation across eutherians remains unclear. In the current study, we measured methylation levels of genes previously identified as methylated in human placenta, in a collection of placental tissue from seven species representing a range of different placenta structure and barrier types indicative of variation across eutherians, including hemomono- and trichorial (primates and mouse respectively), synepitheliochorial (cow) and endotheliochorial (cat).
Our data indicate a conservation of gene methylation within primates (old and new world monkeys and apes). However, the distribution and absolute levels of methylation showed some variation in these closely related species, particularly in the CYP24A1 gene that is more highly methylated in humans relative to baboon and marmoset, and the APC gene that shows an inverse pattern of methylation.
The findings of the current study add a further dimension to processes potentially contributing to differential placental expression, by demonstrating limited conservation of locus-specific methylation patterns, even between closely related species. Little conservation of the methylation profile in APC, SFRP2, DNMT1 or CYP24A1 genes was found in non-primate species, suggesting that these locus-specific methylation events arose relatively recently in evolution. However, some evidence for a reduced level of methylation, or reduced incidence of monoallelic methylation (possibly reflecting differing cellular composition in placental tissue) was evident. This will require further investigation, preferably in purified placenta-derived cell populations.
The consistency of DNA methylation patterns at specific loci within species, and the coordinated methylation of multiple genes within signaling pathways (eg. APC and SFRP2 in primates), implicates genetic factors in the establishment of placental methylation profile. Future studies will be required to identify such factors to fully elucidate the role that such methylation plays in mediating specific aspects of placental development and function.
Finally, the mounting evidence for significant epigenetic discordance between human and animal placentas may limit the value of animal models in unraveling the complex interplay between environment, epigenetic disruption, and phenotype likely to contribute to elevated risk of adverse pregnancy outcomes in humans.
Acknowledgements
We would like to thank Dr Neroli Sunderland (Royal Prince Alfred Hospital, Australia) for full term placental tissue and CVS samples, Drs Patrick Western and Craig Smith (Murdoch Childrens Research Institute, Australia) for mouse placental tissue, Monash Animal Services (Australia) for Marmoset placental tissue, and Dr Chris Thurgood (RSPCA, Victoria) for feline placental tissue. This work was funded by National Health and Medical Research Council (Australia) special grant to JC and RS. SH is a JS Davies Fellow.
Appendix. Supplementary data
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PII: S0143-4004(10)00035-4
doi:10.1016/j.placenta.2010.01.009
© 2010 Elsevier Ltd. All rights reserved.
