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Cultured Cyto and Syncytio-trophoblast samples exposed to varying degrees of hypoxia (expression)

ABSTRACT: The mechanisms by which the placenta adapts to exogenous stimuli to create a stable and healthy environment for the growing fetus are not well known. Low oxygen tension and sub-optimal vitamin D levels influence placental function and both are associated with preeclampsia, a condition associated with altered development of placental trophoblast. We hypothesized that oxygen tension, vitamin D levels, or both affect villous trophoblast by modulation of gene expression through DNA methylation. To test this we used the Illumina Infinium Human Methylation 450 BeadChip array to compare the DNA methylation profile of primary cultures of human cytotrophoblasts and syncytiotrophoblasts under three oxygen tensions and three vitamin D levels. We found no effect on global DNA methylation by either treatment, but a limited set of loci became hypermethylated in cytotrophoblasts exposed for 24 hours to 1% oxygen, as compared to the same cells exposed to 8% or 20% oxygen. Vitamin D levels had no detectable effect on methylation profiles in either trophoblast type. Hypermethylation with low oxygen tension was independently confirmed by bisulfite-pyrosequencing in a subset of functionally important genes including CP, ITGA5, SOD2, XDH and ZNF2. Intriguingly, 70 out of the 147 hypoxia-associated CpGs, overlapped with CpG sites that become hypomethylated upon differentiation of cytotrophoblasts into syncytiotrophoblasts. Furthermore, the preponderance of altered sites was located at AP-1 binding sites. We suggest that AP-1 expression is triggered by hypoxia and interacts with DNA methyltransferases (DNMTs) to target methylation at specific sites in the genome, thus causing suppression of the associated genes that are responsible for differentiation of villous cytotrophoblast to syncytiotrophoblast.

ORGANISM(S): Homo sapiens

PROVIDER: GSE41331 | GEO | 2013/01/18

SECONDARY ACCESSION(S): PRJNA176596

REPOSITORIES: GEO

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Project description:The mechanisms by which the placenta adapts to exogenous stimuli to create a stable and healthy environment for the growing fetus are not well known. Low oxygen tension and sub-optimal vitamin D levels influence placental function and both are associated with preeclampsia, a condition associated with altered development of placental trophoblast. We hypothesized that oxygen tension, vitamin D levels, or both affect villous trophoblast by modulation of gene expression through DNA methylation. To test this we used the Illumina Infinium Human Methylation 450 BeadChip array to compare the DNA methylation profile of primary cultures of human cytotrophoblasts and syncytiotrophoblasts under three oxygen tensions and three vitamin D levels. We found no effect on global DNA methylation by either treatment, but a limited set of loci became hypermethylated in cytotrophoblasts exposed for 24 hours to 1% oxygen, as compared to the same cells exposed to 8% or 20% oxygen. Vitamin D levels had no detectable effect on methylation profiles in either trophoblast type. Hypermethylation with low oxygen tension was independently confirmed by bisulfite-pyrosequencing in a subset of functionally important genes including CP, ITGA5, SOD2, XDH and ZNF2. Intriguingly, 70 out of the 147 hypoxia-associated CpGs, overlapped with CpG sites that become hypomethylated upon differentiation of cytotrophoblasts into syncytiotrophoblasts. Furthermore, the preponderance of altered sites was located at AP-1 binding sites. We suggest that AP-1 expression is triggered by hypoxia and interacts with DNA methyltransferases (DNMTs) to target methylation at specific sites in the genome, thus causing suppression of the associated genes that are responsible for differentiation of villous cytotrophoblast to syncytiotrophoblast. RNA from cytotrophoblast from 2 placentas exposed to 3 different conditions of hypoxia (1%,8%,20%) and treated with 3 levels of vitamin D were run on the Illumina HT-12v4 Expression Array

Project description:The mechanisms by which the placenta adapts to exogenous stimuli to create a stable and healthy environment for the growing fetus are not well known. Low oxygen tension and sub-optimal vitamin D levels influence placental function and both are associated with preeclampsia, a condition associated with altered development of placental trophoblast. We hypothesized that oxygen tension, vitamin D levels, or both affect villous trophoblast by modulation of gene expression through DNA methylation. To test this we used the Illumina Infinium Human Methylation 450 BeadChip array to compare the DNA methylation profile of primary cultures of human cytotrophoblasts and syncytiotrophoblasts under three oxygen tensions and three vitamin D levels. We found no effect on global DNA methylation by either treatment, but a limited set of loci became hypermethylated in cytotrophoblasts exposed for 24 hours to 1% oxygen, as compared to the same cells exposed to 8% or 20% oxygen. Vitamin D levels had no detectable effect on methylation profiles in either trophoblast type. Hypermethylation with low oxygen tension was independently confirmed by bisulfite-pyrosequencing in a subset of functionally important genes including CP, ITGA5, SOD2, XDH and ZNF2. Intriguingly, 70 out of the 147 hypoxia-associated CpGs, overlapped with CpG sites that become hypomethylated upon differentiation of cytotrophoblasts into syncytiotrophoblasts. Furthermore, the preponderance of altered sites was located at AP-1 binding sites. We suggest that AP-1 expression is triggered by hypoxia and interacts with DNA methyltransferases (DNMTs) to target methylation at specific sites in the genome, thus causing suppression of the associated genes that are responsible for differentiation of villous cytotrophoblast to syncytiotrophoblast. DNA from 2 cell types from 5 placentas exposed to 3 different conditions of hypoxia (1%,8%,20%) and treated with 3 levels of vitamin D were bisulfite converted and run on the Illumina HumanMethylation450 BeadChip

Project description:Invasion of cytotrophoblasts into uterine tissues is essential for placental development. To identify molecules regulating trophoblast invasion, mRNA signatures of purified villous (CTB, poor invasiveness) and extravillous (EVT, high invasiveness) trophoblasts isolated from first trimester human placentae and villous explant cultures, respectively, were compared using GeneChip analyses yielding 991 invasion/migration related transcripts. Several genes involved in physiological and pathologic cell invasion, including ADAM-12,-19,-28 as well as Spondin-2, were upregulated in EVT. Pathway prediction analyses identified several functional modules associated with either the invasive or the non-invasive trophoblast phenotype. One of the genes which were downregulated in the invasive mRNA pool, heme oxygenase-1 (HO-1), was selected for functional analyses. Real-time PCR analyses, Western blottting, and immunofluorescene of first trimester placentae and differentiating villous explant cultures demonstrated downregulation of HO-1 in invasive EVT as compared to CTB. Modulation of HO-1 expression in loss-of as well as gain-of function cell models (BeWo and HTR8/SVneo, respectively) demonstrated an inverse relationship of HO-1 expression with trophoblast migration in transwell and wound healing assays. Importantly, HO-1 expression led to an increase in protein levels and activity of the nuclear hormone receptor PPARgamma. Pharmacological inhibition of PPARgamma abrogated the inhibitory effects of HO-1 on trophoblast migration. Collectively, our results demonstrate that gene expression profiling of EVT and CTB can be used to unravel novel regulators of cell invasion. Accordingly, we identify heme oxygenase-1 as a negative regulator of trophoblast motility acting via upregulation of PPARgamma. Experiment Overall Design: To identify genes potentially regulating cell invasion trophoblast cells of early human gestation with distinct invasive properties were profiled. Experiment Overall Design: Distinct gene expression signatures of highly invasive EVT (n = 6) and poorly invasive CTB (n = 5) of different first trimester placentae using Affymetrix U133A GeneChips interrogating >20,000 genes were determined.

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Cultured Cyto and Syncytio-trophoblast samples exposed to varying degrees of hypoxia (expression)

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