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

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

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

HSF1 and HSF3 cooperatively regulate the heat shock response in lizards

<div><p>Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of <i>HSP</i> expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog <i>HSF</i> genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs.</p></div

Aberrantly methylated or expressed genes in Leiomyomas.

<p>A: Venn diagrams show the number of aberrantly hypomethylated or hypermethylated genes in 3 paired samples. B: Venn diagrams show the number of aberrantly upregulated or downregulated genes in 3 paired samples. C: Integration of DNA methylation and mRNA expression data in uterine leiomyoma and adjacent myometrium. The numbers of genes which have both aberrant methylation and aberrant mRNA expression are shown. D: Functional analysis of 24 genes that have aberrant hypomethylation and upregulation of mRNA. The functional categories are shown with the number of included genes and p-values. Functional analysis was performed using IPA. E: Functional analysis of 65 genes that have aberrant hypermethylation and downregulation of mRNA. The functional categories are shown with the number of included genes and p-values. Functional analysis was performed using IPA.</p

Lizard and frog HSF3 as well as HSF1 can induce HSP70 expression.

<p>(<b>A</b>) Induction of HSP70 by lizard HSFs. HSF1-null MEF cells (HSF1-/-) were infected for 48 h with adenovirus expressing GFP, AsHSF1-HA, AsHSF2-HA, AsHSF3-HA, or AsHSF4-HA. These cells and wild-type MEF cells (HSF1+/+) were untreated (C) or treated with heat shock at 42^°C for 1 h and allowed to recover for 3 h (HS + R). Cell extracts were prepared from these cells and aliquots were subjected to western blotting using the indicated antibodies. Positions of molecular weight markers are indicated. Only the lower band of AsHSF3-HA (arrow) was detected by anti-HA antibody. (<b>B</b>) Induction of HSP70 by frog HSFs. HSF1-null cells were infected with adenovirus expressing GFP, XtHSF1-HA, XtHSF2-HA, XtHSF3-HA, or XtHSF4-HA. These cells were treated and analyzed as described in A. (<b>C</b>) Induction of HSP70 by vertebrate HSF1 members. HSF1-null cells were infected with adenovirus expressing GFP, XtHSF1-HA, AsHSF1-HA, cHSF1, or hHSF1, and were treated as described in A. Aliquots of cell extracts were subjected to western blotting using the indicated antibodies including anti-HSF1 (anti-cHSF1x). (<b>D</b>) Induction of HSP70 by vertebrate HSF3 members. HSF1-null cells were infected with adenovirus expressing GFP, XtHSF3-HA, AsHSF3-HA, cHSF3, or mHSF3, and were treated as described in A. Aliquots of cell extracts were subjected to western blotting using the indicated antibodies including anti-HSF3 (anti-XtHSF3-2 or anti-mHSF3-1). (<b>E</b>) Induction of HSP70 mRNA by vertebrate HSF1 and HSF3 members. HSF1-null cells (HSF1-/-) were infected with the indicated adenoviruses for 48 h. These cells and wild-type MEF cells (HSF1+/+) were untreated (C) or treated with heat shock at 42^°C for 30 min (HS). HSP70 mRNA levels were quantified by RT-qPCR are showen (n = 3). *, p < 0.01 versus each control value; **, p < 0.05 versus control value of GFP-expressing cells by Student’s t-test.</p