Aging, Environment, & DiseaseChromatin StructureDevelopmental Biology & Stem Cells

An Epigenetic Link to Recurrent Miscarriage

Epigenetic Link to Recurrent MiscarriageMiscarriage, or natural loss of a pregnancy, is a common reproductive problem and can be upsetting for families trying to have a child. Recurrent miscarriage (RM) – two or more miscarriages – often occur because of problems that arise very early in embryonic development. A common cause of RM is the failure of trophoblasts, the earliest cells of the developing embryo, to properly invade into uterine blood and lymphatic vessels [1]. Trophoblasts eventually develop into placental cells, which are integral for maintaining the connection between a mother and her developing child [2]. Recent work has shown that defects in epigenetic regulation can lead to impaired trophoblast invasion [3, 4]. A new study by Lv and colleagues investigated the role of EZH2 (enhance of zeste homolog 2), a chromatin repressor, on trophoblast invasion in patients with RM.

By comparing villous tissue isolated from healthy women to women who had experienced RM, the authors discovered that patients with RM had lower expression of EZH2 than healthy women. Was the decrease in EZH2 expression related to the decrease in trophoblast invasion often seen in patients with RM? Using JAR cells, which are a model for extravillous trophoblast (EVT) cells, they showed that EZH2 knockdown or inhibition resulted in decreased trophoblast invasion using a transwell assay. The authors observed the inverse effect when EZH2 was over-expressed in JAR cells, which resulted in increased invasion of trophoblasts.

Because they found that EZH2 was involved in trophoblast invasion, the authors wanted to better understand the underlying mechanism. To address this, they looked at how the loss of EZH2 affected the expression of 15 known anti-invasion genes in trophoblast cells. By screening the expression levels of these genes following EZH2 knockdown, they saw that four of the genes had significantly increased expression. Focusing on the two most upregulated genes, CDH1, which encodes the protein E-cadherin, and CDX1, the authors confirmed that EZH2 knockdown in JAR cells resulted in not only increased transcription of these genes, but also increased protein expression. In fact, when they looked in patient samples, the authors also found that CDX1 expression was higher in villous tissue from RM patients than healthy women. Taken together, these results suggest that CDX1 and E-cadherin are involved in the pathogenesis of RM.

Next, the researchers asked if EZH2 represses CDX1 expression by directly binding to its promoter. Using chromatin immunoprecipitation (Ch-IP) the authors showed that EZH2 physically interacts with the CDX1 promoter and that this interaction was also associated with high levels of H3K27me3, a histone methylation marker known for its repressive function on gene expression. Furthermore, using a dual-luciferase reporter assay, the authors found that over-expression of EZH2 resulted in inhibition of CDX1 promoter activity, indicating that EZH2 inhibits CDX1 expression through direct binding to its promoter.

To test if CDX1 acts in conjunction with EZH2 to regulate trophoblast invasion, the authors over-expressed CDX1 in JAR cells and observed decreased trophoblast invasion, consistent with what they observed following EZH2 knockdown. In addition, when over-expression of CDX1 was combined with EZH2 knockdown in the same cells, the authors saw an even greater deficiency in trophoblast invasion, indicating that EZH2 and CDX1 function in the same pathway to influence invasion.

Interestingly, the hormone progesterone has been associated with EZH2 during pregnancy [5]. To see if progesterone affects the expression of EZH2 in trophoblasts, the authors treated JAR cells with progesterone for 24 hours. They observed by Western Blot that EZH2 protein levels increased and those of CDX1 decreased following treatment, suggesting that progesterone regulates EZH2 expression. Because the MEK-ERK1/2-Elk-1 signaling pathway is involved in EZH2 upregulation [6], the authors asked whether progesterone treatment activates this signaling pathway in trophoblasts. In fact, they found that ERK1/2 signaling was activated through an increase in ERK1/2 phosphorylation.  Inhibition of ELK1/2 in progesterone-treated trophoblasts resulted in both a decrease in EZH2 protein levels and in an increase in CDX1 levels, indicating that progesterone upregulates the expression of EZH2 through the ERK1/2 pathway.

Altogether, this work elucidates the underlying mechanism behind defects in trophoblast invasion in women with recurrent miscarriage. A decrease in EZH2 and a corresponding increase in CDX1 and E-cadherin is associated with decreased trophoblast invasion. Because treatment with progesterone increased EZH2 expression, progesterone may be an important candidate for treatment of recurrent miscarriage, thus providing hope to many wishing to grow their family.

 

References:

Original article: Lv S, Wang N, Lv H, Yang J, Liu J, Li WP, Zhang C, Chen ZJ (2018). The Attenuation of Trophoblast Invasion Caused by the Downregulation of EZH2 Is Involved in the Pathogenesis of Human Recurrent Miscarriage. Mol Ther Nucleic Acids, 14:377-387. doi: 10.1016/j.omtn.2018.12.011.

[1] Windsperger K, Dekan S, Pils S, Golletz C, Kunihs V, Fiala C, Kristiansen G, Knöfler M, Pollheimer J (2017). Extravillous trophoblast invasion of venous as well as lymphatic vessels is altered in idiopathic, recurrent, spontaneous abortions. Human Reprod, 32(6):1208-1217. doi: 10.1093/humrep/dex058.

[2] Renaud SJ, Soares MJ (2011). Chapter 77 – Strategies for investigating hemochorial placentation. Reproductive and Developmental Toxicology, Academic Press: 1029-1038. https://doi.org/10.1016/B978-0-12-382032-7.10077-3

[3] Novakovic B, Gordon L, Wong NC, Moffett A, Manuelpillai U, Craig JM, Sharkey A, Saffery R (2011). Wide-ranging DNA methylation differences of primary trophoblast cell populations and derived cell lines: implications and opportunities for understanding trophoblast function. Mol Hum Reprod, 17(6):344-53. doi: 10.1093/molehr/gar005.

[4] Kohan-Ghadr HR, Kadam L, Jain C, Armant DR, Drewlo S (2016). Potential role of epigenetic mechanisms in regulation of trophoblast differentiation, migration, and invasion in the human placenta. Cell Adh Migr, 10(1-2):126-35. doi: 10.1080/19336918.2015.1098800.

[5] Pal B, Bouras T, Shi W, Vaillant F, Sheridan JM, Fu N, Breslin K, Jiang K, Ritchie ME, Young M, Lindeman GJ, Smyth GK, Visvader JE (2013). Global changes in the mammary epigenome are induced by hormonal cues and coordinated by Ezh2. Cell Rep, 3(2):411-26. doi: 10.1016/j.celrep.2012.12.020.

[6] Fujii S, Tokita K, Wada N, Ito K, Yamauchi C, Ito Y, Ochiai A (2011). MEK-ERK pathway regulates EZH2 overexpression in association with aggressive breast cancer subtypes. Oncogene, 30(39):4118-28. doi: 10.1038/onc.2011.118.

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Stephanie DeMarco

Stephanie DeMarco

Stephanie is a PhD candidate in Molecular Biology at the University of California, Los Angeles where she studies how the parasite Trypanosoma brucei regulates its social behavior. When she’s not wrangling her parasites in the lab, Stephanie likes to write about science, tap dance, and attempt to make the perfect plate of pasta carbonara.