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.