Analysis of Genomic DNA Methylation Levels in Human Placenta using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

Putra, Sulistyo Emantoko Dwi; Neuber, Corinna; Reichetzeder, Christoph; Hocher, Berthold; Kleuser, Burkhard

doi:10.1159/000358666

Cited by 17 publications

(12 citation statements)

References 26 publications

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“…Therefore, one of the ways in which LAQ824 treatment improved the in vitro developmental competence of porcine SCNT embryos may be that increased histone acetylation levels, caused by promotion of HATs and inhibition of HDAC activities, may facilitate chromatin opening. DNA methylation, as another epigenetic marker [43], also plays an important role in the process of reprogramming and influences in vitro developmental competence of SCNT embryos via the activity of DNA methyltransferases (DNMTs). In this study, we revealed that LAQ824 treatment downregulated the mRNA expression of DNMT1, 3a and 3b in blastocysts.…”

Section: Discussionmentioning

confidence: 99%

The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos

Jin

Lee

Taweechaipaisankul

et al. 2017

Cell Physiol Biochem

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Background/Aims: Hypoacetylation caused by aberrant epigenetic nuclear reprogramming results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic remodeling drugs have been used in attempts to improve in vitro development of porcine SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone acetylase inhibitor, on the nuclear reprogramming and in vitro development of porcine SCNT embryos. Methods: LAQ824 treatment was supplemented during the culture of SCNT embryos. The reprogramming levels were measured by immunofluorescence and quantified by image J software. Relative expression levels of 18 genes were analyzed by quantitative real-time PCR. Results: 100 nM LAQ824 treatment of post-activation SCNT embryos for 24 h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression of genes for histone acetylation (HAT1, HDAC1, 2, 3, and 6), DNA methylation (DNMT1, 3a and 3b), development (Pou5f1, Nanog, Sox2, and GLUT1) and apoptosis (Bax, Bcl2, Caspase 3 and Bak) in blastocysts. Conclusion: Optimum exposure (100 nM for 24 h) to LAQ824 post-activation improved the in vitro development of porcine SCNT embryos by enhancing levels of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.

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Section: Discussionmentioning

confidence: 99%

The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos

Jin

Lee

Taweechaipaisankul

et al. 2017

Cell Physiol Biochem

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“…Seventy microliters of the hydrolised DNA samples were further diluted with 280 μL 0.1 % formic acid to yield a final concentration of 2 ng digested DNA/μL. DNA methylation was assessed by liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS) technique as described previously [ 56 ]. LC-ESI/MS/MS was performed with an Agilent 1200 series HPLC system connected to an Agilent 6530 Accurate-Mass Q-TOF instrument with Jet Stream-Interface (Waldbronn, Germany).…”

Section: Methodsmentioning

confidence: 99%

Increased global placental DNA methylation levels are associated with gestational diabetes

et al. 2016

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BackgroundGestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM.MethodsGlobal DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study.ResultsMothers with GDM displayed a significantly increased global placental DNA methylation (3.22 ± 0.63 vs. 3.00 ± 0.46 %; p = 0.013; ±SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1–4 (2.9–5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation.ConclusionsThis is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.

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“…1982 , Fuke et al . 2004 ), a large study in 248 placentas has shown that there is a large range of DNA methylation of 2–5% ( Dwi Putra et al . 2014 ), but the reason for this variation requires further investigation.…”

Section: Foetal Sex Differences In Dna Methylationmentioning

confidence: 99%

Recent progress towards understanding the role of DNA methylation in human placental development

Bianco‐Miotto¹,

Mayne²,

Buckberry

et al. 2016

Reproduction

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Epigenetic modifications, and particularly DNA methylation, have been studied in many tissues, both healthy and diseased, and across numerous developmental stages. The placenta is the only organ that has a transient life of 9 months and undergoes rapid growth and dynamic structural and functional changes across gestation. Additionally, the placenta is unique because although developing within the mother, its genome is identical to that of the foetus. Given these distinctive characteristics, it is not surprising that the epigenetic landscape affecting placental gene expression may be different to that in other healthy tissues. However, the role of epigenetic modifications, and particularly DNA methylation, in placental development remains largely unknown. Of particular interest is the fact that the placenta is the most hypomethylated human tissue and is characterized by the presence of large partially methylated domains (PMDs) containing silenced genes. Moreover, how and why the placenta is hypomethylated and what role DNA methylation plays in regulating placental gene expression across gestation are poorly understood. We review genome-wide DNA methylation studies in the human placenta and highlight that the different cell types that make up the placenta have very different DNA methylation profiles. Summarizing studies on DNA methylation in the placenta and its relationship with pregnancy complications are difficult due to the limited number of studies available for comparison. To understand the key steps in placental development and hence what may be perturbed in pregnancy complications requires large-scale genome-wide DNA methylation studies coupled with transcriptome analyses.

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Analysis of Genomic DNA Methylation Levels in Human Placenta using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

Cited by 17 publications

References 26 publications

The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos

The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos

Increased global placental DNA methylation levels are associated with gestational diabetes

Recent progress towards understanding the role of DNA methylation in human placental development

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