DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter

Integrative analysis of methylome and transcriptome in human blood identifies extensive sex- and immune cell-specific differentially methylated regions

<div><p>The relationship between DNA methylation and gene expression is complex and elusive. To further elucidate these relations, we performed an integrative analysis of the methylome and transcriptome of 4 circulating immune cell subsets (B cells, monocytes, CD4⁺, and CD8⁺ T cells) from healthy females. Additionally, in light of the known sex bias in the prevalence of several immune-mediated diseases, the female datasets were compared with similar public available male data sets. Immune cell-specific differentially methylated regions (DMRs) were found to be highly similar between sexes, with an average correlation coefficient of 0.82; however, numerous sex-specific DMRs, shared by the cell subsets, were identified, mainly on autosomal chromosomes. This provides a list of highly interesting candidate genes to be studied in disorders with sexual dimorphism, such as autoimmune diseases. Immune cell-specific DMRs were mainly located in the gene body and intergenic region, distant from CpG islands but overlapping with enhancer elements, indicating that distal regulatory elements are important in immune cell specificity. In contrast, sex-specific DMRs were overrepresented in CpG islands, suggesting that the epigenetic regulatory mechanisms of sex and immune cell specificity may differ. Both positive and, more frequently, negative correlations between subset-specific expression and methylation were observed, and cell-specific DMRs of both interactions were associated with similar biological pathways, while sex-specific DMRs were linked to networks of early development or estrogen receptor and immune-related molecules. Our findings of immune cell- and sex-specific methylome and transcriptome profiles provide novel insight on their complex regulatory interactions and may particularly contribute to research of immune-mediated diseases.</p></div

Methylome and transcriptome profiling in Myasthenia Gravis monozygotic twins

To identify novel genetic and epigenetic factors associated with Myasthenia gravis (MG) using an identical twins experimental study design.The transcriptome and methylome of peripheral monocytes were compared between monozygotic (MZ) twins discordant and concordant for MG, as well as with MG singletons and healthy controls, all females. Sets of differentially expressed genes and differentially methylated CpGs were validated using RT-PCR for expression and target bisulfite sequencing for methylation on additional samples.&gt;100 differentially expressed genes and ∼1800 differentially methylated CpGs were detected in peripheral monocytes between MG patients and controls. Several transcripts associated with immune homeostasis and inflammation resolution were reduced in MG patients. Only a relatively few genes differed between the discordant healthy and MG co-twins, and both their expression and methylation profiles demonstrated very high similarity.This is the first study to characterize the DNA methylation profile in MG, and the expression profile of immune cells in MZ twins with MG. Results suggest that numerous small changes in gene expression or methylation might together contribute to disease. Impaired monocyte function in MG and decreased expression of genes associated with inflammation resolution could contribute to the chronicity of the disease. Findings may serve as potential new predictive biomarkers for disease and disease activity, as well as potential future targets for therapy development. The high similarity between the healthy and the MG discordant twins, suggests that a molecular signature might precede a clinical phenotype, and that genetic predisposition may have a stronger contribution to disease than previously assumed

Methylome and transcriptome profiling in Myasthenia Gravis monozygotic twins.

OBJECTIVE: To identify novel genetic and epigenetic factors associated with Myasthenia gravis (MG) using an identical twins experimental study design. METHODS: The transcriptome and methylome of peripheral monocytes were compared between monozygotic (MZ) twins discordant and concordant for MG, as well as with MG singletons and healthy controls, all females. Sets of differentially expressed genes and differentially methylated CpGs were validated using RT-PCR for expression and target bisulfite sequencing for methylation on additional samples. RESULTS: >100 differentially expressed genes and approximately 1800 differentially methylated CpGs were detected in peripheral monocytes between MG patients and controls. Several transcripts associated with immune homeostasis and inflammation resolution were reduced in MG patients. Only a relatively few genes differed between the discordant healthy and MG co-twins, and both their expression and methylation profiles demonstrated very high similarity. INTERPRETATION: This is the first study to characterize the DNA methylation profile in MG, and the expression profile of immune cells in MZ twins with MG. Results suggest that numerous small changes in gene expression or methylation might together contribute to disease. Impaired monocyte function in MG and decreased expression of genes associated with inflammation resolution could contribute to the chronicity of the disease. Findings may serve as potential new predictive biomarkers for disease and disease activity, as well as potential future targets for therapy development. The high similarity between the healthy and the MG discordant twins, suggests that a molecular signature might precede a clinical phenotype, and that genetic predisposition may have a stronger contribution to disease than previously assumed

<i>Oxtr</i> mRNA levels correlate with <i>Oxtr</i> promoter methylation in cell lines.

<p><b>A</b>) The relative mRNA levels in GT1-7 are significantly higher than in 4T1 cells. <b>B</b>) Methylation of the seven CpG sites is higher in 4T1 cells than in GT1-7 cells. Each row represents a single clone and each column represents one of the seven CpG sites. The total percentage of methylation was calculated from the fraction of black spots (methylated CpG sites). <b>C</b>) Representative gel showing greater ERK phosphorylation in GT1-7 cells stimulated with 1 µM OT for 10 min compared vehicle-treated cells Graph is summary of three independent experiments. <b>D</b>) Quantities of <i>Oxtr</i> mRNA in GT1-7 cells following 24 h treatment with 1 µM OT. An OT-stimulated increase in <i>Oxtr</i> mRNA was documented in three independent experiments. <b>Statistics</b> (t-test): <b>A</b>) *<i>p</i><0.001, n = 4. <b>C</b>) *<i>p</i><0.05, n = 3. <b>D</b>) *<i>p</i><0.01 n = 3.</p

Methylation of specific CpG sites in the <i>Oxtr</i> minimal promoter inhibits transcription.

<p><b>A</b>) A schematic depiction of the distinct <i>Oxtr</i> promoter/<i>EYFP</i> constructs used; An <i>EYFP</i> gene was coupled to a minimal promoter (positions −1417 to +46) of the mouse <i>Oxtr</i> gene (<i>Unmodified</i>). This construct was modified by a C to A mutation at CpG sites 1 (<i>Mut 1</i>) or site 7 (<i>Mut 7</i>) or by deleting the ∼400 bp amplicon region (<i>Del</i>). <b>B,C</b>) EYFP mean fluorescence intensity measured in GT1-7 cells that were transfected with either (<b>B</b>) untraeatd or (<b>C</b>) methyltransferase-treated <i>Oxtr</i> promoter/<i>EYFP</i> constructs. No significant differences were found among the untreated plasmids whereas a highly significant difference was found among the methyltransferase-treated plasmids. <b>D</b>) A comparison between the treatedand untreated versions of all plasmids. Highly significant differences were found only in the cases of the <i>Unmodified</i> and <i>Mut 1</i> plasmids. <b>Statistics</b>: <b>B</b>) one-way ANOVA F₍₃₎ = 2.32, <i>p</i>>0.1. <b>C</b>) - one-way ANOVA F₍₃₎ = 9.4, <i>p</i><0.01, * differ from <i>Unmodified</i>, <i>p</i><0.05, n = 5. <b>D</b>) t-test, *<i>p</i><0.05/4.</p

The general structure of the mouse <i>Oxtr</i> gene and its promoter.

<p><b>A</b>) A schematic depiction of the mouse <i>Oxtr</i> gene, including the CpG island that extends from the promoter into the coding region at the third exon. <b>B</b>) A schematic depiction of the EREs and SP1 binding sites in the minimal gene promoter, and the relative location of the PCR amplicon between positions −956 and −541. <b>C</b>) The sequence of the PCR amplicon, including the positions of the seven CpG sites. The ERE harboring CpG site 1 and the SP1 binding sequence harboring CpG site 7 are underlined.</p

PCR primer sets.

<p>T_A, Temperatures and time applied for annealing.</p

<i>Oxtr</i> promoter methylation changes <i>in vivo</i>.

<p><b>A</b>) The methylation pattern of the <i>Oxtr</i> promoter shows no significant difference between the mammary glands (black bars, n = 3) and uterus (gray bars, n = 4) of virgin mice. Nevertheless, a significant difference in methylation was found between CpG sites among all animals. <b>B</b>) <i>Oxtr</i> mRNA levels in the mammary glands (black bars, n = 4) and uterus (gray bars, n = 5) are significantly elevated 1 h following parturition (early postpartum). Whereas in the uterus mRNA levels dropto background 24 h later (late postpartum, n = 3), they remain high in the mammary glands (n = 2). <b>C</b>) The mean difference in the methylation of the seven examined CpG sites, between early postpartum and virgin females. A general increase is observed in the mammary glands while a general decrease is evident in the uterus. <b>D</b>) In contrast to virgin mice a statistically significant difference in <i>Oxtr</i> promoter methylation is observed between the mammary glands (black bars) and uterus (gray bars) of early post-partum mice. These differences are found in CpG sites 3–7. <b>E</b>) The mean relative methylation level of the <i>Oxtr</i> promoter, averaged over CpG sites 3–7, is decreased in the uterus in early postpartum and returns to baseline 24 h later. In contrast, in the mammary glands it remains high during late postpartum. <b>F</b>) The mean relative methylation level of the <i>Oxtr</i> promoter, plotted as a function of the <i>Oxtr</i> expression in all animals. A statistically significant positive correlation is seen in the mammary glands (black rectangles), compared to a negative correlation in the case of the uterus (gray circles). <b>Statistics</b>: <b>A</b>) Between conditions - repeated two-way ANOVA F_(6,30), between groups <i>p</i>>0.2, interaction between factors <i>p</i>>0.4. Between CpG sites - repeated one-way ANOVA F₍₆₎ = 20.9, <i>p</i><0.001, * differ from all others <i>p</i><0.05. <b>B</b>) Uterus - one-way ANOVA F₍₂₎ = 10.1, <i>p</i><0.005, * and † differ from virgins, <i>p</i><0.01. Mammary glands - one-way ANOVA F₍₂₎ = 19.6, <i>p</i><0.005, * differ from virgins <i>p</i><0.01. <b>D</b>) Repeated one-way ANOVA F(1) = 6.0, <i>p</i><0.05. <b>E</b>) Uterus - one-way ANOVA F₍₂₎ = 50.6, <i>p</i><0.001, * differ virgins <i>p</i><0.01. Mammary glands - one-way ANOVA F₍₂₎ = 11.4, <i>p</i><0.005, † differ from all others <i>p</i><0.01). <b>F</b>) Mammary glands - Pearson correlation, <i>p</i><0.01, R² = 0.61. Uterus - Pearson correlation, <i>p</i><0.05, R² = 0.48.</p