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

Hypomethylation in choESC compred to euESCa.

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

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

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

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

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

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

Average methylation profiles (beta values) obtained for the CpG-s analysed in each experimental condition.

<p>Each density plot represents the median of three samples.</p

High-content image analysis of the heterogeneous population of SIRT1 immunostained cells of the four groups suggests changes in the cell nucleus.

<p><b>A.</b> Examples of images used for the analysis of SIRT1 expression representative of 9 experiments. The groups and colour codes are the same as indicated on the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048943#pone-0048943-g001" target="_blank">Figure 1</a>. Scale = 50 micrometers. Confocal analysis was performed on a LEICA TCS SP2 microscope (Leica Microsystems) with the Leica Confocal Software. Images were acquired with a 40X HCX PL APO Plan Fluor oil objective (NA 1.25) at room temperature. For display, the images were processed using ImageJ a median filter 3 pixels followed by an image adjustment, increasing the brightness was performed equally on all the Red images. The nuclei were stained with DAPI and the SIRT1 antibody was labelled with Alexa 594 <b>B.</b> Upper plot: A representative example of the principal component analysis of the data extracted from the SIRT1 immunostaining images. Individual cells are visualized as points on the scatter plot of the first two principal components describing the variation. They were identified by the colour code according to their group of origin only after their position on the scatter plot is calculated. Note that cells from the same experimental group tend to cluster. The groups 2 and 3 overlap. Lower plot: Representative example of the LDA analysis of the same data. This alternative multiparametric analysis takes into account the existence of four groups and helps to visualize the differences between them. Note the overall similarity of the LDA and PCA scatter plots.</p

X chromosomal microsatellite analysis of leiomyoma and adjacent myometrium.

<p>Chromatographs using four tandem repeat markers (DXS7132, DXS6789, DXS8377 and DXS6807) are indicated. Chromatographs of DNA from leiomyoma (L) and adjacent myometrium (M) obtained from Case1 (Top), Case2 (Middle) and Case3 (Bottom) are shown. The PCR products of DXS7132, DXS6789, DXS8377 and DXS6807 were separated electrophoretically on a Fluorescent Capillary System ABI PRISM 310 and analyzed with GeneScan software (Applied Biosystems). The allelic status in each case was examined by comparing the allelic statuses of the leiomyoma and adjacent myometrium.</p