Down-regulation of the histone methyltransferase EZH2 contributes to the epigenetic programming of decidualizing human endometrial stromal cells

Abstract

Differentiation of human endometrial stromal cells (HESC) into decidual cells represents a highly coordinated process essential for embryo implantation. We show that decidualizing HESC down-regulate the histone methyltransferase enhancer of Zeste homolog 2 (EZH2), resulting in declining levels of trimethylation of histone 3 on lysine 27 (H3K27me3) at the proximal promoters of key decidual marker genes PRL and IGFBP1. Loss of H3K27me3 was associated with a reciprocal enrichment in acetylation of the same lysine residue, indicating active remodeling from repressive to transcriptionally permissive chromatin. Chromatin immunoprecipitation coupled with DNA microarray analysis demonstrated that decidualization triggers genome-wide changes in H3K27me3 distribution that only partly overlap those observed upon EZH2 knockdown in undifferentiated HESC. Gene ontology revealed that gain of the repressive H3K27me3 mark in response to decidualization and upon EZH2 knockdown in undifferentiated cells was enriched at the promoter regions of genes involved in transcriptional regulation and growth/cell proliferation, respectively. However, loss of the H3K27me3 mark (indicating increased chromatin accessibility) in decidualizing cells and upon EZH2 knockdown occurred at selective loci enriched for genes functionally implicated in responses to stimulus. In agreement, EZH2 knockdown in undifferentiated HESC was sufficient to augment the induction of decidual marker genes in response to cyclic AMP and progesterone signaling. Thus, loss of EZH2-dependent methyltransferase activity in the endometrium is integral to the process of chromatin remodeling that enables the transition from a proliferative to a decidual phenotype in response to differentiation cues.

Fig. 1.

Cycle-dependent expression of EZH2 in human endometrium. A, Protein lysates from proliferative (PE) and secretory (SE) endometrium were subjected to Western blot analysis and immunoprobed for EZH2. β-Actin served as a loading control. Panel on the right shows densitometric analysis of Western blots using ImageJ. B, EZH2 transcript levels in timed endometrial biopsies obtained during the early secretory (ES) (n = 5), midsecretory (MS) (n = 5), and late secretory (LS) (n = 5) phases of the cycle were determined by RT-qPCR. The data, normalized to L19 mRNA, are expressed in arbitrary units (a.u.); mean ± sem. C, Endometrial tissue sections obtained at different phases of the cycle were immunostained for EZH2 (brown). The data shown in this figure are representative of three or more independent experiments.

Fig. 2.

EZH2 is down-regulated upon decidualization. A, Confluent HESC cultures were either untreated (d 0) or decidualized with 8-Br-cAMP and MPA for the indicated time points. EZH2 expression at mRNA and protein levels were determined in parallel cultures by RT-qPCR (upper panel) and Western blot analysis (lower panel). The RT-qPCR data show the fold change (±sem of triplicate measurements) in EZH2 transcript levels upon treatment relatively to expression in untreated cells. B, EZH2 expression in endometrial stromal cells cultured in the presence or absence of 8-Br-cAMP and MPA for 2, 4, and 8 d. The intensity of EZH2 (red channel) and DAPI nuclear staining (blue channel) were captured by confocal microscopy. C, Confluent HESC cultures either untreated or decidualized with 8-Br-cAMP (cAMP), MPA, or a combination for 2 d. EZH2 expression at mRNA and protein levels were determined in parallel cultures by RT-qPCR (upper panel) and Western blot analysis (lower panel). The data show the fold change (±sem of triplicate measurements) in EZH2 transcript levels upon treatment relatively to expression in untreated cells. a.u., Arbitrary units.

Fig. 3.

H3K27 methylation in undifferentiated and decidualizing HESC. A, Primary HESC treated with 8-Br-cAMP and MPA for the indicated time points were fixed in formaldehyde, stained, and subjected to confocal microscopy. The upper panel represents H3K27me3 staining (red channel), whereas the lower panel shows the corresponding nuclear DAPI staining (blue channel). B, Total protein lysates of HESC treated as above were subjected to Western blot analysis and immunoprobed for EZH1, H3K27me, and total H3. β-Actin was used as a loading control. C, Chromatin extracted from HESC first decidualized for 2, 4, or 8 d was immunoprecipitated with antibodies against H3K27me3 and IgG. The chromatin, normalized to the input, was analyzed by qPCR (ChIP-qPCR) with primers specific for the promoter regions of PRL (left panel) and IGFBP1 (right panel). Data are expressed as the fold change (±sem of triplicate measurements) relatively to the abundance of chromatin-bound H3K27me3 in untreated cells. The data shown in this figure are representative of three independent experiments.

Fig. 4.

Dynamic changes in H3K27 acetylation upon decidualization of HESC. A, Endometrial stromal cells were cultured in the presence or absence of 8-Br-cAMP and MPA for the indicated time points. The intensity of H3K27ac (green channel) and the corresponding DAPI nuclear staining (blue channel) were captured by confocal microscopy. B, Whole-cell protein lysates of HESC treated as above were subjected to Western blot analysis using a H3K27ac-specific antibody. β-Actin served as a loading control. C, ChIP was carried out on HESC decidualized for the indicated time points using antibodies against H3K27ac and IgG. qPCR was carried out on the ChIPed DNA with primers specific for the proximal promoter regions of PRL (left panel) and IGFBP1 (right panel) and normalized to the input. Data are expressed as the fold change (±sem of triplicate measurements) relatively to the abundance of chromatin-bound H3K27ac in untreated cells. The data shown in this figure are representative of three independent experiments.

Fig. 5.

EZH2 down-regulation is permissive for the induction of decidual marker genes. A, Total cell lysates from primary HESC cultures first transfected with either NTor EZH2 siRNA and then treated with cAMP and MPA for 2 d were subjected to Western blot analysis and probed with EZH1-, EZH2-, H3K27me3-, and H3-specific antibodies. β-Actin served as a loading control. B, ChIP with H3K27me3 and IgG antibodies was performed on cultures treated as above. The precipitated DNA fragments were analyzed by qPCR and normalized to the input. The primers used were specific for the proximal promoter regions of IGFBP1 (left panel) and PRL (right panel). Data are expressed as the fold change (±sem of triplicate measurements) relatively to the abundance of chromatin-bound H3K27me3 in untreated cells. C, ChIP was carried out with H3K27ac-specific antibody under the experimental conditions described above. D, PRL and IGFBP-1 transcript levels were measured by RT-qPCR in HESC first transfected with either NT or EZH2 siRNA and then treated with cAMP and MPA for 2 d. The data show fold-change (±sem of triplicate measurements) in expression relative to untreated cells transfected with NT siRNA. E, Protein lysates extracted from cells first transfected with an empty control vector (pCMV) or a plasmid encoding EZH2 (pCMV-EZH2) vector and then treated with decidualization stimuli for 2 d were subjected to Western blot analysis and immunoprobed for EZH2. β-Actin was used as a loading control. F, Total RNA from parallel cultures was examined for the expression of IGFBP-1 (left panel) and PRL (right panel) transcripts by RT-qPCR. The data (mean ± sem) are presented as fold induction, and the results are representative of three repeat experiments.

Fig. 6.

Genome-wide redistribution of H3K27me3 in decidualizing HESC and upon EZH2 knockdown in undifferentiated HESC. A, Venn diagram indicating the number of genomic loci significantly (P < 0.05) altered in H3K27 methylation as determined by ChIP-chip analysis of HESC either decidualized with 8-Br-cAMP and MPA for 8 d (left) or upon EZH2 knockdown (right). B, Example of altered H3K27me3 peaks in the genomic region encompassing the IGFBP1 promoter. The blue line represents the peaks in the undifferentiated cells, whereas the red line indicates the corresponding relative level of H3K27me3 in paired cultures decidualized with 8-Br-cAMP and MPA for 8 d. C, GO analysis of genes in the proximity of genomic loci characterized by significant loss (bottom panel) or gain (top panel) in H3K27me3 upon decidualization of HESC. D, GO enrichment analysis of promoter regions altered in H3K27 methylation in response to differentiation as well as siRNA-mediated EZH2 knockdown in undifferentiated cultures.