Global Analyses of the Placental Epigenome in Preeclampsia

项目摘要/摘要 我们推测胎盘表观基因组及其与转录组的关系是 了解在重度子痫前期(SPE)发病机制中具有重要作用的通路。这 假说是基于SPE与某些胎盘病理的关联。细胞滋养层细胞 侵犯子宫壁不能正确区分；CTB侵犯蜕膜较浅， 血管内侵袭受到限制。最近我们发现，光滑绒毛的CTB也有很大的 与SPE相关的显著的形态和分子变化。受影响妊娠的绒毛 也有明显的异常，如合胞体结节。这项提案的调查人员-专家在 表观基因组分析、生物统计学和生物信息学、数据可视化和人类胎盘生物学- 完成了详细的转录和表观基因组图谱，在正常妊娠的第二和第三个三个月， 在SPE-CTB中被破坏的区域中，光滑的绒毛和绒毛。全基因组亚硫酸氢盐 测序(WGBS)证实了胎盘DNA的低甲基化，并首次显示出大的 低甲基化的阻断标志着抑制H3K9me3的进展。DNA甲基化的模式是 对于每个样本类型和三个月都是唯一的，建议动态调节。随着孕期的推进，许多 CTB基因组的调节区变得甲基化，表明表观遗传机制调节 功能改变。对相应的RNA-seq数据的分析表明，CTB的转录产物是 在妊娠中期高表达，在足月下调的基因包括更多在 SPE比预期的要好。令人兴奋的免疫印迹(IB)数据，由免疫组织化学证实， 从胎盘中分离出的CTB在组蛋白修饰水平上显示出新的和强烈的差异 被诊断为SPE的妇女和对照样本，与胎龄匹配，从 无感染迹象的早产妇女的胎盘(NPTL)。我们从理论上认为这种融合 来自CTB的表观基因组和转录组数据，SPE中光滑的绒毛和绒毛将揭示 不规范的路径和新的机械洞察力。至于方法，我们将使用WGBS来分析DNA 甲基化(目标1)。我们将使用IB和CHIP-SEQ来评估组蛋白修饰-H3K27me3，H3k9me3， H3K4me1、H3K4me3和H3K27ac(目标2)。此外，我们还将通过以下方式探索这些发现的翻译潜力 询问是否可以在母体中检测到组蛋白异常修饰的SPE相关图谱 血浆。我们将应用rna-seq在mrna水平上研究表观遗传改变的后果。 并通过体外结核功能检测来检验研究结果的意义(目标3)。结果将是 可通过WASHU Eigenome浏览器公开获取。因此，我们的结果将揭示 表观基因组在SPE相关胎盘基因表达的变化和该疾病的候选生物标记物。

PROJECT SUMMARY/ABSTRACT We theorize that the placental epigenome and its relationship to the transcriptome hold the key to understanding pathways with important roles in the pathogenesis of severe preeclampsia (sPE). This hypothesis is based on the association of sPE with certain placental pathologies. The cytotrophoblasts (CTBs) that invade the uterine wall fail to differentiate properly; CTB invasion of the decidua is shallow and endovascular invasion is constrained. Recently we found that CTBs of the smooth chorion also have very significant sPE-associated morphological and molecular changes. Chorionic villi from affected pregnancies have overt abnormalities as well such as syncytial knots. The investigators on this proposal—experts in epigenomic analyses, biostatistics and bioinfomatics, data visualization and human placental biology— completed detailed transcriptomic and epigenomic profiling, in the 2nd and 3rd trimesters of normal pregnancy, of the areas that are disrupted in sPE—CTBs, the smooth chorion and chorionic villi. Whole genome bisulfite sequencing (WGBS) confirmed hypomethylation of placental DNA and showed, for the first time, that large blocks of hypomethylation were marked with gains in repressive H3K9me3. Patterns of DNA methylation were unique to each sample type and trimester, suggesting dynamic regulation. As gestation advanced, many regulatory regions of the CTB genome became methylated, suggesting epigenetic mechanisms regulating functional alterations. Analyses of the corresponding RNA-seq data showed that CTB transcripts that were highly expressed in 2nd trimester and downregulated at term included more genes that are overexpressed in sPE than would be expected by chance. Exciting immunoblot (IB) data, corroborated by immunohistochemistry, showed a novel and strong difference in histone modification levels between CTBs isolated from the placentas of women diagnosed with sPE and control samples, matched for gestational age, that were isolated from the placentas of women who had a preterm birth with no sign of infection (nPTL). We theorize that coalescing epigenomic and transcriptomic data from CTBs, the smooth chorion and chorionic villi in sPE will reveal the dysregulated pathways and new mechanistic insights. As to approach, we will use WGBS to profile DNA methylation (Aim 1). We will employ IB and ChIP-seq to assess histone modifications—H3k27me3, H3k9me3, H3K4me1, H3K4me3 and H3K27ac (Aim 2). Also, we will explore the translational potential of the findings by asking whether the sPE-associated profile of dysregulated histone modifications can be detected in maternal plasma. We will apply RNA-seq to investigate the consequences of epigenetic alterations at the mRNA level and test the significance of the findings by using in vitro assays of TB functions (Aim 3). Results will be publically available through the WashU Epigenome Browser. Thus, our results will reveal the role of the epigenome in sPE-related changes in placental gene expression and candidate biomarkers of this condition.