Down-Regulation of the Histone Methyltransferase EZH2 Contributes to the Epigenetic Programming of Decidualizing Human Endometrial Stromal Cells (original) (raw)
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Expression of epigenetic effectors in decidualizing human endometrial stromal cells
Molecular Human Reproduction, 2012
Cyclic differentiation of human endometrial stromal cells (HESCs) into decidual cells is a highly coordinated process essential for embryo implantation and pregnancy. This differentiation process is closely recapitulated in culture upon exposure of purified HESCs to cyclic AMP and progesterone signaling. Mining of gene expression data revealed that HESCs express 147 genes coding for epigenetic effectors, 33 (22%) of which are significantly regulated (P , 0.05) upon decidualization. Among these are genes encoding for histone-modifying proteins and their cofactors, histone-binding proteins, histone variants, CpG-binding proteins and DNA methyltransferases (DNMTs). Interestingly, more than two-thirds of differentially expressed chromatin-modifying genes are down-regulated upon the transition from a proliferative to a differentiated HESC phenotype. Despite the strong regulation of DNMTs, colorimetric and long interspersed nuclear element 1 methylation assays did not show global changes in DNA methylation levels upon differentiation of HESCs. Taken together, the coordinated regulation of diverse effector molecules suggests that complex epigenetic modification at specific loci underpins the acquisition of a decidual endometrial phenotype.
Epigenetic Changes Through DNA Methylation Contribute to Uterine Stromal Cell Decidualization
Endocrinology, 2012
Embryo-uterine interaction during early pregnancy critically depends on the coordinated expression of numerous genes at the site of implantation. The epigenetic mechanism through DNA methylation (DNM) plays a major role in the control of gene expression, although this regulatory event remains unknown in uterine implantation sites. Our analysis revealed the presence of DNA methyltransferase 1 (Dnmt1) in mouse endometrial cells on the receptive d 4 of pregnancy and early postattachment (d 5) phase, whereas Dnmt3a had lower abundant expression. Both Dnmt1 and Dnmt3a were coordinately expressed in decidual cells on d 6 -8. 5-Methycytosine showed a similar expression pattern to that of Dnmt1. The preimplantation inhibition of DNM by 5-aza-2Ј-deoxycytodine was not antagonistic for embryonic attachment, although endometrial stromal cell proliferation at the site of implantation was down-regulated, indicating a disturbance with the postattachment decidualization event. Indeed, the peri-or postimplantation inhibition of DNM caused significant abrogation of decidualization, with concomitant loss of embryos. We next identified decidual genes undergoing alteration of DNM using methylation-sensitive restriction fingerprinting. One such gene, Chromobox homolog 4, an epigenetic regulator in the polycomb group protein family, exhibited hypomethylation in promoter DNA and increased expression with the onset of decidualization. Furthermore, inhibition of DNM resulted in enhanced expression of hypermethylated genes (Bcl3 and Slc16a3) in the decidual bed as compared with control, indicating aberration of gene expression may be associated with DNM-inhibition-induced decidual perturbation. Overall, these results suggest that uterine DNM plays a major role for successful decidualization and embryo development during early pregnancy. (Endocrinology 153: 6078 -6090, 2012) E mbryo implantation is a complex process that requires two-way interactions between developmentally competent blastocysts and a receptive uterus. Proper implantation is essential for continued embryonic development within the uterus and achieving successful pregnancy (1, 2). In mice, the embryonic attachment reaction occurs in the evening (2200 -2300 h) on d 4 of pregnancy (d 1, vaginal plug) and coincides with increased localized vascular permeability in the endometrium, which can be visualized by discrete blue reaction along the uterus after an iv injection of a blue dye solution (3). The attachment reaction is followed by localized stromal cell transformation into decidual cells (decidualization) and luminal ep-ithelial apoptosis at the sites of blastocyst implantation. The decidualization process begins with extensive stromal cell proliferation in the morning on d 5, followed by regional differentiation into specialized cells (decidual cells) with acquisition of polyploidy on d 6 -8 (4 -6). Decidual cells at the antimesometrial pole initially form the primary decidual zone in the afternoon on d 5, which is avascular and epithelioid in nature . From d 5 afternoon through d 6, stromal cells next to the primary decidual zone continue to proliferate and differentiate into polyploid decidual cells, forming the secondary decidual zone. The secondary decidual zone is fully developed by d 7 afternoon, and at this time, polyploidy development gradually
MHR: Basic science of reproductive medicine, 2012
Progesterone, estrogen and cyclic adenosine monophosphate (cAMP) together regulate the decidualization of human endometrial stromal cells in a time-dependent manner. The role of DNA methylation and the three active DNA methyltransferases (DNMTs) in the regulation of decidualization is gaining interest but the exact role of this epigenetic mechanism during decidualization is largely unknown. We aimed to understand the effect of the main regulators of decidualization on the expression of the DNMTs and in turn on the expression of steroid hormone receptors during the decidualization. We conducted a time-course analysis from 6 h to 10 days to examine the change in gene expression of the DNMTs and the steroid hormone receptors over time in response to estradiol, medroxy-progesterone acetate (MPA) and dibutyryl-cAMP (db-cAMP) in a human endometrial stromal cells (HESC) cell line. Only the combination treatment with MPA-mix (estradiol + MPA + db-cAMP) up-regulated ERa, PGR, progesterone receptor B (PRB) and androgen receptor at 24 h. Both decidualization pathways of db-cAMP and estradiol/MPA, independently and combined, consistently down-regulated DNMT3B mRNA expression from 6 h till 10 days, whereas DNMT1 and DNMT3A mRNA expression were down-regulated transiently. Forced expression of DNMT3B in HESC for 10 days attenuated IGFBP1 mRNA and protein expression; and forced expression of DNMT3B combined with MPA-mix treatment synergistically increased the expression of PRB at 24 h. The HESC morphology and proliferation remained unchanged in response to forced expression of DNMT3B. In conclusion, mRNA expression of the DNMTs during decidualization is dynamic, so that expression varies according to the cAMP or estradiol/MPA pathway treatments that regulate them in a time-dependent manner. Although forced expression of DNMT3B by itself is insufficient to inhibit decidualization, forced expression of DNMT3B in combination with MPA-mix synergistically upregulated PRB, as well as attenuated the expression of IGFBP1, the decidualization marker.
Analysis of chromatin accessibility in decidualizing human endometrial stromal cells
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018
Spontaneous decidualization of the endometrium in response to progesterone signaling is confined to menstruating species, including humans and other higher primates. During this process, endometrial stromal cells (EnSCs) differentiate into specialized decidual cells that control embryo implantation. We subjected undifferentiated and decidualizing human EnSCs to an assay for transposase accessible chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility upon decidualization was strongly associated with differential gene expression. Analysis of 1533 opening and closing chromatin regions revealed over-representation of DNA binding motifs for known decidual transcription factors (TFs) and identified putative new regulators. ATAC-seq footprint analysis provided evidence of TF binding at specific motifs. One of the largest footprints involved the most enriched motif-basic...
The Roles of the Histone Protein Modifier EZH2 in the Uterus and Placenta
Epigenomes
Epigenetic modifications regulate normal physiological, as well as pathological processes in various organs, including the uterus and placenta. Both organs undergo dramatic and rapid restructuring that depends upon precise orchestration of events. Epigenetic changes that alter transcription and translation of gene-sets regulate such responses. Histone modifications alter the chromatin structure, thereby affecting transcription factor access to gene promoter regions. Binding of histones to DNA is regulated by addition or removal of subunit methyl and other groups, which can inhibit or stimulate transcription. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2) that catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) and subsequently suppresses transcription of genes bound by such histones. Uterine EZH2 expression exerts a critical role in development and function of this organ with deletion of this gene resulting in uterine hyp...
Changes in global gene expression during in vitro decidualization of rat endometrial stromal cells
Journal of Cellular Physiology, 2010
During the preimplantation phase of pregnancy the endometrial stroma differentiates into decidua, a process that implies numerous morphological changes and is an example of physiological transdifferentiation. Here we show that UIII rat endometrial stromal cells cultured in the presence of calf serum acquired morphological features of decidual cells and expressed decidual markers. To identify genes involved in decidualization we compared gene expression patterns of control and decidualized UIII cells using cDNA microarray. We found 322 annotated genes exhibiting significant differences in expression (>3-fold, fold discovery rate (FDR) >0.005), of which 312 have not been previously related to decidualization. Analysis of overrepresented functions revealed that protein synthesis, gene expression, and chromatin architecture and remodeling are the most relevant modified functions during decidualization. Relevant genes are also found in the functional terms differentiation, cell proliferation, signal transduction, and matrix/structural proteins. Several of these new genes involved in decidualization (Csdc2, Trim27, Eef1a1, Bmp1, Wt1, Aes, Gna12, and Men1) are shown to be also regulated in uterine decidua during normal pregnancy. Thus, the UIII cell culture model will allow future mechanistic studies to define the transcriptional network regulating reprogramming of stromal cells into decidual cells.
DNA Methylation Regulates Human Endometrial Stromal Cell Decidualisation
2012
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EZH2 Is Required for Uterine Epithelial Integrity
The American Journal of Pathology
Normal proliferation and differentiation of uterine epithelial cells are critical for uterine development and function. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a core component of polycomb repressive complexes 2, possesses histone methyltransferase activity that catalyzes the trimethylation of lysine 27 of histone H3. EZH2 has been involved in epithelial-mesenchymal transition, a key event in development and carcinogenesis. However, its role in uterine epithelial cell function remains unknown. To determine the role of uterine EZH2, Ezh2 was conditionally deleted using progesterone receptor Cre recombinase, which is expressed in both epithelial and mesenchymal compartments of the uterus. Loss of EZH2 promoted stratification of uterine epithelium, an uncommon and detrimental event in the uterus. The abnormal epithelium expressed basal cell markers, including tumor protein 63, cytokeratin 5 (KRT5), KRT6A, and KRT14. These results suggest that EZH2 serves as a guardian of uterine epithelial integrity, partially via inhibiting the differentiation of basal-like cells and preventing epithelial stratification. The observed epithelial abnormality was accompanied by fertility defects, altered uterine growth and function, and the development of endometrial hyperplasia. Thus, the Ezh2 conditional knockout mouse model may be useful to explore mechanisms that regulate endometrial homeostasis and uterine function.
Physiological Genomics, 2020
Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that suppresses gene expression. Previously, we developed a conditional null model where EZH2 is knocked out in uterus. Deletion of uterine EZH2 increased proliferation of luminal and glandular epithelial cells. Herein, we used RNA-Seq in wild-type (WT) and EZH2 conditional knockout (Ezh2cKO) uteri to obtain mechanistic insights into the gene expression changes that underpin the pathogenesis observed in these mice. Ovariectomized adult Ezh2cKO mice were treated with vehicle (V) or 17-estradiol (E2; 1 ng/g). Uteri were collected at postnatal day (PND) 75 for RNA-Seq or immunostaining for epithelial proliferation. Weighted gene coexpression network analysis was used to link uterine gene expression patterns and epithelial proliferation. In V-treated mice, 88 transcripts were differentially expressed (DEG) in Ezh2cKO mice, and Bmp5, Crabp2, Lgr5, and Sprr2f were upregulated. E2 treatment resulted in 40 DEG with Krt5, Krt15, Olig3, Crabp1, and Serpinb7 upregulated in Ezh2cKO compared with control mice. Transcript analysis relative to proliferation rates revealed two module eigengenes correlated with epithelial proliferation in WT V vs. Ezh2cKO V and WT E2 vs. Ezh2cKO E2 mice, with a positive relationship in the former and inverse in the latter. Notably, the ESR1, Wnt, and Hippo signaling pathways were among those functionally enriched in Ezh2cKO females. Current results reveal unique gene expression patterns in Ezh2cKO uterus and provide insight into how loss of this critical epigenetic regulator assumingly contributes to uterine abnormalities.
Biomolecular concepts, 2014
DNA methylation at cytosines is an important epigenetic modification that participates in gene expression regulation without changing the original DNA sequence. With the rapid progress of high-throughput sequencing techniques, whole-genome distribution of methylated cytosines and their regulatory mechanism have been revealed gradually. This has allowed the uncovering of the critical roles played by DNA methylation in the maintenance of cell pluripotency, determination of cell fate during development, and in diverse diseases. Recently, rediscovery of 5-hydroxymethylcytosine, and other types of modification on DNA, have uncovered more dynamic aspects of cell methylome regulation. The interaction of DNA methylation and other epigenetic changes remodel the chromatin structure and determine the state of gene transcription, not only permanently, but also transiently under certain stimuli. The uterus is a reproductive organ that experiences dramatic hormone stimulated changes during the es...