Genome-Wide DNA Methylation Profiling in Cultured Eutopic and Ectopic Endometrial Stromal Cells

<div><p>The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa) and without endometriosis (euESCb) and ovarian endometrial cysts (choESC). Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2), which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.</p></div

Volksrecht : Sozialdemokratisches Tagblatt für die politischen Bezirke Aussig und Leitmeritz.

The "Volksrecht" is a socialist newspaper for the political districts of Aussig (Ústí nad Labem) and Leitmeritz (Litoměřice).Electronic reproduction.Description based on: Jahrgang 25, Nr. 276 (1. Dez. 1920); caption title.Latest issue consulted: Jahrgang 25, Nr. 300 (31. Dez. 1920

Hypomethylation in choESC compred to euESCa.

<p>Hypomethylation in choESC compred to euESCa.</p

Hypermethylation in choESC compred to euESCa.

<p>Lists of statistically significant GO terms (Biological process and molecular function) and KEGG pathway terms in hypomethylated genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083612#pone-0083612-t001" target="_blank">Table 1</a>) and in hypermethylated genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083612#pone-0083612-t002" target="_blank">Table 2</a>) in choESC compared to euESCa.</p

The DNA methylation status as determined by the methylation-sensitive high resolution analyses (MS-HRMA) of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in euESCa and choESC.

<p>Sample No.-wide DNA methylation and transpciptome as euESCa, and sample No. 8, 9, 10 were also analyzed as choESC. Sample No. 1 and 10 were analyzed for bisulfite sequencing as euESCa and choESC, respectively. The confidence value calculated by MS-HRMA indirectly indicates the DNA methylation level. The DNA methylation status of euESCa-1 was shown as 100% identical with regard to the DNA methylation. The confidence values of each sample were calculated in comparison with euESCa-1. In NR5A1 and STAR, the confidence values were lower in choESC than those in euESCa, indicating that the choESC are hypomethylated compared with the euESCa. In STRA6 and HSD17B2, the DNA methylation status of euESCa-1 was shown as −100% (arbitrary defined reverse axis value) identical regarding DNA methylation, indicating a DNA hypomethylation status. In STRA6, the confidence values were higher in choESC than those in euESCa, indicating that the choESC are hypermethylated compared with the euESCa. In HSD17B2, the DNA methylation status varied among individuals with euESCa and choESC. The samples of euESCa and choESC were isolated from seven and six patients, respectively.</p

The mRNA levels of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in ESCa, choESC, eutopic endometrium and chocolate cysts.

<p>ESCa (n = 7), choESC (n = 5), eutopic endometria (n = 17) and chocolate cysts (n = 6) were subjected to total RNA isolation followed by real-time RT-PCR. The relative mRNA expression normalized to that of TBP (an internal control) was calculated. The values are the means ±SD. *, <i>p</i><0.01. ND: not detected.</p

Effects of H₂O₂ on HSR and expression of scavenger enzymes.

<p><i>A</i>) Total RNA was harvested from T98G cells at the indicated hours after the indicated treatments (44°c for 20 min and/or 0.25 mM H₂O₂) and transcripts of the indicated genes were evaluated by RT-PCR. <i>B</i>) Confocal microscopical detection of HSP70. Cells were pretreated with 0.25 mM H₂O₂ (H₂O₂) or untreated (control) and fixed at 2 or 4 h after heat exposure. HSP70 expression was immuunohistochemically detected using the anti-HSP70 antibody and cells were counter-stained with propidium iodide (PI). Bar indicates 10 µm. <i>C</i>) eIF2α and JNK phosphorylation. At 1 and 5 h after indicated treatments as described above, eIF2α and JNK activities were evaluated by western blots using anti-phosphorylated eIF2α and anti-phosphorylated JNK antibodies, respectively. HSP70 and catalase expression levels were also evaluated. Anti-β-actin, anti-eIF2α and anti-JNK protein antibodies show equal loading of protein samples.</p

The results of the sodium bisulfite sequencing analyses of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in the euESCa and choESC samples.

<p>The DNA methylation profile in the genomic regions of the NR5A1, STAR, STRA6 and HSD17B2 genes was analyzed by a sodium bisulfite sequencing method in a pair of euESCa and choESC from one individual, which had already been analyzed by the Infinium method. In NR5A1 and STRA6, the DNA methylation status of the proximal promoter and first exon was analyzed. The primer pairs BP-A and BP-B amplify region A and B, respectively, in STRA6. In STAR, the distal promoter region was analyzed. In HSD17B2, the first intron and second exon region were analyzed. The arrows indicate the positions of the bisulfite primers. Closed triangles represent the CpG sites analyzed by the Infinium method, and are accompanied by the identification names. •, methylated CpG sites; ◯, unmethylated CpG sites; BP, bisulfite primer.</p

Recovery of protein folding activity.

<p><i>A</i>) Refolding activity. Cells were transiently co-transfected with the pGRE/RL-TK reporter genes, treated with 5 mM dexamethazone for 10-12 h, exposed to heat (44°c for 20 min) with (closed) or without (open bars) 0.25 mM H₂O₂ pretreatment, incubated for 2 to 4 h and luciferase activity was measured. Cells were also incubated with 5 mM L-NAC for 30 min prior to 0.25 mM H₂O₂ pretreatment. *, <i>P</i><0.05, **, <i>P</i><0.01 compared with H₂O₂-untreated cells. <i>B</i>) HSP70 transcripts. Total RNA was harvested from cells treated as indicated and HSP70 transcripts were evaluated by RT-PCR (left). HSP70 transcripts were determined by real-time PCR and normalized to GAPDH levels (right panel). **, <i>P</i><0.01 compared with heat/H₂O₂-treated cells. Columns display the mean ± S.D. of data from three separate experiments.</p

Effect of H₂O₂ on heat-exposed HSF +/+ and -/- MEFs.

<p><i>A</i>) Cell death at 24 h after heat exposure (42.5°c for the indicated min) with (closed) or without (open bars) 0.5 mM H₂O₂ pretreatment. <i>B</i>) HSP70 transcripts evaluated by RT-PCR transcription at the indicated hours after the indicated treatments (upper) were determined by real-time PCR with normalization to GAPDH levels (lower panel). <i>C</i>) Preconditioning effect on disruption of Δψm. Both MEF cells were treated as indicated, heat (Heat; 42.5°c for 20 min), 0.5 mM H₂O₂ treatment (H₂O₂) and both treatments (Both). As thermal preconditioning, cells were preheated (40.5°c for 30 min 10 h) prior to both treatments (Preheat/Both). Cells were cultured for 20 h after heat exposure and incubated with DePsipher solution. Numbers indicate % of cells showing loss of Δψm. <i>D</i>) Effect of H₂O₂ pretreatment on refolding activity. Refolding activity was evaluated by recovery of luciferase activity 5 h after heat exposure (42.5°c for 20 min) with (closed) or without (open bars) 0.5 mM H₂O₂ pretreatment. Cells were also treated by thermal preconditioning (Preheat) as described in <i>C</i>. In <i>A</i>, <i>B</i> and <i>D</i>, columns display the mean ± S.D. of data from three separate experiments and *, <i>P</i><0.05; **, <i>P</i><0.01 compared with H₂O₂-untreated cells.</p