Cell specific patterns of methylation in the human placenta

Epigenetic processes, such as DNA methylation, are known to regulate tissue specific gene expression. We explored this concept in the placenta to define whether DNA methylation is cell-type specific. Cytotrophoblasts and fibroblasts were isolated from normal midtrimester placentas. Using immunocytochemistry, we demonstrated 95% purity for cytotrophoblasts and 60-70% for fibroblasts. We compared DNA methylation profiles from cytotrophoblasts, fibroblasts and whole placental villi using bisulfite modified genomic DNA hybridized to the Illumina Methylation27 array. Euclidean cluster analysis of the DNA methylation profiles showed 2 main clusters, one containing cytotrophoblasts and placenta, the other fibroblasts. Differential methylation analysis identified 442 autosomal CpG sites that differed between cytotrophoblasts and fibroblasts, 315 between placenta and fibroblasts and 61 between placenta and cytotrophoblasts. Three candidate methylation differences were validated by targeted pyrosequencing assays. Pyrosequencing assays were developed for CpG sites less methylated in cytotrophoblasts than fibroblasts mapping to the promoter region of the beta subunit of human chorionic gonadotropin 5 (CGB5), as well as 2 CpG sites mapping to each of 2 tumor suppressor genes. Our data suggest that epigenetic regulation of gene expression is likely to be a key factor in the functional specificity of cytotrophoblasts. These data are proof of principle for cell-type specific epigenetic regulation in placenta and demonstrate that the methylation profile of placenta is mainly driven by cytotrophoblasts.     

Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum

Interspecific hybridization is known for triggering genetic and epigenetic changes, such as modifications on DNA methylation patterns and impact on phenotypic plasticity and ecological adaptation. Wild potatoes (Solanum, section Petota) are adapted to multiple habitats along the Andes, and natural hybridizations have proven to be a common feature among species of this group. Solanum × rechei, a recently formed hybrid that grows sympatrically with the parental species S. kurtzianum and S. microdontum, represents an ideal model for studying the ecologically and evolutionary importance of hybridization in generating of epigenetic variability. Genetic and epigenetic variability and their correlation with morphological variation were investigated in wild and ex situ conserved populations of these three wild potato species using amplified fragment length polymorphism (AFLP) and methylation‐sensitive amplified polymorphism (MSAP) techniques. We observed that novel methylation patterns doubled the number of novel genetic patterns in the hybrid and that the morphological variability measured on 30 characters had a higher correlation with the epigenetic than with the genetic variability. Statistical comparison of methylation levels suggested that the interspecific hybridization induces genome demethylation in the hybrids. A Bayesian analysis of the genetic data reveled the hybrid nature of S. × rechei, with genotypes displaying high levels of admixture with the parental species, while the epigenetic information assigned S. × rechei to its own cluster with low admixture. These findings suggested that after the hybridization event, a novel epigenetic pattern was rapidly established, which might influence the phenotypic plasticity and adaptation of the hybrid to new environments.     

Fine‐scale population epigenetic structure in relation to gastrointestinal parasite load in red grouse (Lagopus lagopus scotica)

Epigenetic modification of cytosine methylation states can be elicited by environmental stresses and may be a key process affecting phenotypic plasticity and adaptation. Parasites are potent stressors with profound physiological and ecological effects on their host, but there is little understanding in how parasites may influence host methylation states. Here, we estimate epigenetic diversity and differentiation among 21 populations of red grouse (Lagopus lagopus scotica) in north‐east Scotland and test for association of gastrointestinal parasite load (caecal nematode Trichostrongylus tenuis) with hepatic genome‐wide and locus‐specific methylation states. Following methylation‐sensitive AFLP (MSAP), 129 bands, representing 73 methylation‐susceptible and 56 nonmethylated epiloci, were scored across 234 individuals. The populations differed significantly in genome‐wide methylation levels and were also significantly epigenetically (F_SC = 0.0227; P < 0.001) and genetically (F_SC = 0.0058; P < 0.001) differentiated. Parasite load was not associated with either genome‐wide methylation levels or epigenetic differentiation. Instead, we found eight disproportionately differentiated epilocus‐specific methylation states (F_ST outliers) using bayescan software and significant positive and negative association of 35 methylation states with parasite load from bespoke generalized estimating equations (GEE), simple logistic regression (sam ) and Bayesian environmental analysis (bayenv 2). Following Sanger sequencing, genome mapping and geneontology (go ) annotation, some of these epiloci were linked to genes involved in regulation of cell cycle, signalling, metabolism, immune system and notably rRNA methylation, histone acetylation and small RNAs. These findings demonstrate an epigenetic signature of parasite load in populations of a wild bird and suggest intriguing physiological effects of parasite‐associated cytosine methylation.     

Lentiviral Transduction of CD34+ Cells Induces Genome-Wide Epigenetic Modifications

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34⁺ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34⁺ cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34⁺ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.     

Methylation differences of the neuronatin gene promoter region in liver between normal and cloned pigs

Abnormal phenotypes in cloned pigs can be partly due to changes in epigenetic modifications such as methylation levels of promoter CpG islands. Neuronatin is an imprinted gene, conserved in human, pig, cattle and mouse, which is expressed exclusively from the paternal allele. Three CpG islands located in the promoter region of the porcine neuronatin gene have the potential to regulate the gene expression by cytosine methylation. To illustrate whether neuronatin was differentially expressed among nuclear transfer macroglossia–positive and nuclear transfer macroglossia–negative pigs and in vitro‐fertilized pigs, we detected its expression level by qRT‐PCR and further quantified methylation levels by pyrosequencing DNA from the liver. The results showed that neuronatin was expressed at a significantly higher level in livers of nuclear transfer macroglossia‐positive pigs compared with normal cloned and in vitro‐fertilized pigs. Livers of nuclear transfer macroglossia‐positive pigs also had a significantly lower methylation level at CpG island 2 and CpG island 3 in the promoter region.     

Cell specific patterns of methylation in the human placenta

Epigenetic processes, such as DNA methylation, are known to regulate tissue specific gene expression. We explored this concept in the placenta to define whether DNA methylation is cell-type specific. Cytotrophoblasts and fibroblasts were isolated from normal midtrimester placentas. Using immunocytochemistry, we demonstrated 95% purity for cytotrophoblasts and 60–70% for fibroblasts. We compared DNA methylation profiles from cytotrophoblasts, fibroblasts and whole placental villi using bisulfite modified genomic DNA hybridized to the Illumina Methylation27 array. Euclidean cluster analysis of the DNA methylation profiles showed two main clusters, one containing cytotrophoblasts and placenta, the other fibroblasts. Differential methylation analysis identified 442 autosomal CpG sites that differed between cytotrophoblasts and fibroblasts, 315 between placenta and fibroblasts and 61 between placenta and cytotrophoblasts. Three candidate methylation differences were validated by targeted pyrosequencing assays. Pyrosequencing assays were developed for CpG sites less methylated in cytotrophoblasts than fibroblasts mapping to the promoter region of the beta subunit of human chorionic gonadotropin 5 (CGB5), as well as two CpG sites mapping to each of two tumor suppressor genes. Our data suggest that epigenetic regulation of gene expression is likely to be a key factor in the functional specificity of cytotrophoblasts. These data are proof of principle for cell-type specific epigenetic regulation in placenta and demonstrate that the methylation profile of placenta is mainly driven by cytotrophoblasts.Key words: cytotrophoblast purification, placental fibroblast purification, DNA methylation, epigenetics, placenta, cell type-specific methylation     

Senescence‐associated DNA methylation is stochastically acquired in subpopulations of mesenchymal stem cells

Replicative senescence has a major impact on function and integrity of cell preparations. This process is reflected by continuous DNA methylation (DNAm) changes at specific CpG dinucleotides in the course of in vitro culture, and such modifications can be used to estimate the state of cellular senescence for quality control of cell preparations. Still, it is unclear how senescence‐associated DNAm changes are regulated and whether they occur simultaneously across a cell population. In this study, we analyzed global DNAm profiles of human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) to demonstrate that senescence‐associated DNAm changes are overall similar in these different cell types. Subsequently, an Epigenetic‐Senescence‐Signature, based on six CpGs, was either analyzed by pyrosequencing or by bar‐coded bisulfite amplicon sequencing. There was a good correlation between predicted and real passage numbers in bulk populations of MSCs (R² = 0.67) and HUVECs (R² = 0.97). However, when we analyzed the Epigenetic‐Senescence‐Signature in subclones of MSCs, the predictions revealed high variation and they were not related to the adipogenic or osteogenic differentiation potential of the subclones. Notably, in clonally derived subpopulations, the DNAm levels of neighboring CpGs differed extensively, indicating that these genomic regions are not synchronously modified during senescence. Taken together, senescence‐associated DNAm changes occur in a highly reproducible manner, but they are not synchronously co‐regulated. They rather appear to be acquired stochastically—potentially evoked by other epigenetic modifications.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Ethnic diversity of DNA methylation in the OPRM1 promoter region in lymphocytes of heroin addicts

The μ-opioid receptor is the site of action of many endogenous opioids as well as opiates. We hypothesize that differences in DNA methylation of specific CpG dinucleotides between former severe heroin addicts in methadone maintenance treatment and control subjects will depend, in part, upon ethnicity. DNA methylation analysis of the μ-opioid receptor gene (OPRM1) promoter region was performed on African-Americans (118 cases, 80 controls) and Hispanics (142 cases, 61 controls) and these were compared with a similar Caucasian cohort from our earlier study. In controls, a higher methylation level was found in the African-Americans compared with the Hispanics or Caucasians. Significant experiment-wise differences in methylation levels were found at the −25 and +12 CpG sites in the controls among the three ethnicities. The overall methylation level of the CpG sites were significantly higher in the former heroin addicts when compared with the controls (point-wise P = 0.0457). However, in the African-Americans, the degree of methylation was significantly decreased experiment-wise in the former heroin addicts at the +12 CpG site (P = 0.0032, Bonferroni corrected general estimating equations). In Hispanics, the degree of methylation was increased in the former heroin addicts at the −25 (P < 0.001, experiment-wise), −14 (P = 0.001, experiment-wise), and +27 (P < 0.001, experiment-wise) CpG sites. These changes in methylation of the OPRM1 promoter region may lead to altered expression of the μ-opioid receptor gene in the lymphocytes of former heroin addicts who are stabilized in methadone maintenance treatment.     

DNA methylation patterns associate with genetic and gene expression variation in HapMap cell lines

Background  

DNA methylation is an essential epigenetic mechanism involved in gene regulation and disease, but little is known about the mechanisms underlying inter-individual variation in methylation profiles. Here we measured methylation levels at 22,290 CpG dinucleotides in lymphoblastoid cell lines from 77 HapMap Yoruba individuals, for which genome-wide gene expression and genotype data were also available.     

Genome‐wide DNA methylation signatures of infection status in Trinidadian guppies (Poecilia reticulata)

Epigenetic modification, especially DNA methylation, can play an important role in mediating gene regulatory response to environmental stressors and may be a key process affecting phenotypic plasticity and adaptation. Parasites are potent stressors with profound physiological and ecological effects on their hosts, yet it remains unclear how parasites influence host methylation patterns. Here, we used a well‐studied host–parasite system, the guppy Poecilia reticulata and its ectoparasitic monogenean Gyrodactylus turnbulli to gain mechanistic insight into the dynamics of DNA methylation in host–parasite interactions. To explore this, we quantitatively measured genome‐wide DNA methylation in guppy skin tissue using reduced representation bisulphite sequencing and characterized differential methylation patterns in guppies during distinct phases of infection. We identified 365, 313, and 741 differentially methylated regions (DMRs) between infected and control fish in early infection, peak infection and recovery phases, respectively. The magnitude of the methylation difference was moderate in DMRs, with an average of 29% (early infection), 27% (peak infection) and 30% (recovery) differential methylation per DMR. Approximately 50% of DMRs overlapped with CpG islands, and over half of the DMRs overlapped with gene bodies, several of which encode proteins relevant to immune response. These findings provide the first evidence of an epigenetic signature of infection by ectoparasites and demonstrate the changing relationship between epigenetic variation and immune response in distinct phases of infection.     

TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.     

DNA methylation pattern of CALCA in preterm neonates with bacterial sepsis as a putative epigenetic biomarker

Diagnosis of bacterial sepsis in preterm neonates can be difficult when using serum markers that rely on physiological changes because these changes may not necessarily be the result of bacterial infections alone. This retrospective investigation explores the potential use of the DNA methylation pattern of CpG sites in the promoter region of the calcitonin-related polypeptide α (CALCA) gene as an epigenetic biomarker for bacterial sepsis in preterm newborns. Four novel changes in the DNA methylation of eight CpG sites were detected in this gene and are present only in neonates with bacterial sepsis: (1) partial methylation at -769 CpG in gram-negative or gram-positive early onset sepsis (EOS) and late onset sepsis (LOS) episodes; (2) demethylation of 8 CpGs in gram-negative EOS followed by LOS (ELS) and in gram-negative EOS; (3) demethylation of 7 CpGs in gram-positive ELS and gram-positive EOS; (4) -771 C:G > T:A; 5′ de novo -778 CpG mutation on both alleles in EOS. These changes were not detected in birth weight and gestational age matched controls or in newborns with isolated infections. Our results indicate that the DNA methylation pattern of the promoter region of the CALCA gene varies in different types of bacterial preterm sepsis, thus suggesting a potential use as an epigenetic biomarker. A prospective confirmation of these results is essential.