H3K27me3 is an Epigenetic Mark of Relevance in Endometriosis

Abstract

Epigenetic mechanisms may play an important role in the etiology of endometriosis. The modification of histones by methylation of lysine residues has been shown to regulate gene expression by changing chromatin structure. We have previously shown that endometriotic lesions had aberrant levels of histone acetylation (lower) and methylation (higher) than control tissues. We aimed to determine the levels of trimethylated histone 3 at lysine residue 27 (H3K27me3), a well-known repressive mark, by immunoassay of fresh tissues and immunohistochemistry (IHC) of an endometriosis-focused tissue microarray. Also, we aimed to determine levels of expression of enhancer of zeste homolog 2 (EZH2), the enzyme responsible for trimethylation of H3K27me3, in cell lines. Average levels of H3K27me3 measured by immunoassay were not significantly different in lesions compared to endometrium from patients and controls. However, there was a trend of higher levels of H3K27me3 in secretory versus proliferative endometrium. The results of IHC showed that lesions (ovarian, fallopian, and peritoneal) and secretory endometrium from controls have higher percentage of H3K27me3-positive nuclei than eutopic endometrium from patients. Endometriotic epithelial cells express high levels of EZH2, which is upregulated by progesterone. This study provides evidence in support of a role of H3K27me3 in the pathogenesis of endometriosis and for EZH2 as a potential therapeutic target for this disease, but more studies are necessary to understand the molecular mechanisms at play.

Keywords: H3K27me3; endometriosis; epigenetics; histone methylation.

Figure 1.

Trimethylation levels in histone 3 lysine (K) residue 27 (H3KK27me3) in endometriosis and endometrium from patients and controls. H3K27me3 levels were determined using the EpiQuick Tri-methylation Assay Kits (Epigentek, Brooklyn, New York) following the manufacturer’s protocols. The amount of methylated protein in nanograms per milligrams of total protein was obtained dividing the methylated protein amount and the total protein input amount divided by the slope. Average total trimethylation levels at H3K27me3 were not significantly different (P = .6273) in endometriotic lesions as compared to eutopic endometrium from patients and from controls. Endometriotic lesions (n = 14), endometrium from women with endometriosis (n = 17), and endometrium from controls (n = 15).

Figure 2.

Representative images (×40) of trimethylation of lysine residue 27 at histone 3 (H3K27me3) showing different staining intensities in glands and stroma. Weak (A), moderate (B), and strong intensity (C) of H3K27me3, respectively, are shown. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)

Figure 3.

Intensity of Trimethylation of lysine residue 27 at histone 3 (H3K27me3) nuclear immunostaining in endometrial and endometriotic samples. A, H3K27me3 nuclear immunostaining in stroma. A total of 147 of 164 formalin-fixed paraffin-embedded endometrial and endometriotic tissues on a tissue microarray were analysed by immunohistochemistry (IHC). B, H3K27me3 nuclear immunostaining in glands. A total of 130 of 164 formalin-fixed paraffin-embedded endometrial and endometriotic tissues on a tissue microarray were analyzed by IHC. Using the Image Scope Program and Aperio Analysis, the immunostaining intensity of H3K27me3 in both compartments (ie, stroma and glands) was evaluated. One-way analysis of variance (ANOVA) was carried out to compare percentage of positive nuclei in endometriotic lesions, proliferative endometrium from patients (cases), and proliferative and secretory endometrium from controls, respectively.

Figure 4.

Immunostaining intensity (HistoScore) of H3K27me3 in endometrial and endometriotic tissues. A total 147 of 164 formalin-fixed paraffin-embedded endometrial and endometriotic tissues on a tissue microarray were analysed by immunohistochemistry (IHC). HistoScore for all types of lesions was shown in (A) and (B). Intensities staining for lesions taking together as a group were showed for stroma (A) and glands (B). One-way analysis of variance (ANOVA) was carried out among endometriotic lesion locations and the proliferative endometrium from patients and proliferative and secretory endometrium from controls. Analysis was conducted separately for stroma and glands.

Figure 5.

Expression of enhancer of zeste homolog 2 (EZH2) on epithelial endometriotic cells (12Z) and nonendometriotic epithelial cells (EEC/MCF7) with ovarian steroid hormones. EZH2 HMT for H3K27me3 was detected at ≈ 75 to 100 kDa. Total protein of hormone-treated cells (10 µg/µL) was extracted and subjected to Western blot using EZH2 polyclonal antibody. Cells were treated with estrogen (E2), progesterone (P4), and E2/P4 at 24 hours. The concentration of E2 used was 1 × 10⁻⁸. P4 concentration used was 1 × 10⁻⁷. Internal control was GAPDH ≈ 37 kDa. Relative densitometry is shown for 3 independent experiments for the 24-hour time point, optical density (OD) units were normalized against GAPDH and presented relative to basal.