Preeclampsia is the major pregnancy-induced hypertensive disorder. It modifies the expression profile of placental genes, including several serine protease inhibitors ( SERPINs ). The objective of this study was to perform a systematic expression analysis of these genes in normal and pathological placentas and to pinpoint epigenetic alterations inside their promoter regions. Expression of 18 placental SERPINs was analyzed by quantitative RT-PCR on placentas from pregnancies complicated by preeclampsia, intrauterine growth restriction, or both and was compared with normal controls. SERPINA3, A5, A8, B2, B5 , and B7 presented significant differences in expression in ≥1 pathological situation. In parallel, the methylation status of the CpG islands located in their promoter regions was studied on a sample of control and preeclamptic placentas. Ten SERPIN promoters were either totally methylated or totally unmethylated, whereas SERPINA3, A5 , and A8 presented complex methylation profiles. For SERPINA3 , the analysis was extended to 81 samples and performed by pyrosequencing. For the SERPINA3 CpG island, the average methylation level was significantly diminished in preeclampsia and growth restriction. The hypomethylated CpGs were situated at putative binding sites for developmental and stress response (hypoxia and inflammation) factors. Our results provide one of the first observations of a specific epigenetic alteration in human placental diseases and provide new potential markers for an early diagnosis.

MHC class II (MHCII) genes are transactivated by the NOD-like receptor (NLR) family member CIITA, which is recruited to SXY enhancers of MHCII promoters via a DNA-binding “enhanceosome” complex. NLRC5, another NLR protein, was recently found to control transcription of MHC class I (MHCI) genes. However, detailed understanding of NLRC5’s target gene specificity and mechanism of action remained lacking. We performed ChIP-sequencing experiments to gain comprehensive information on NLRC5-regulated genes. In addition to classical MHCI genes, we exclusively identified novel targets encoding non-classical MHCI molecules having important functions in immunity and tolerance. ChIP-sequencing performed with Rfx5^−/− cells, which lack the pivotal enhanceosome factor RFX5, demonstrated its strict requirement for NLRC5 recruitment. Accordingly, Rfx5-knockout mice phenocopy Nlrc5 deficiency with respect to defective MHCI expression. Analysis of B cell lines lacking RFX5, RFXAP, or RFXANK further corroborated the importance of the enhanceosome for MHCI expression. Although recruited by common DNA-binding factors, CIITA and NLRC5 exhibit non-redundant functions, shown here using double-deficient Nlrc5^−/−CIIta^−/− mice. These paradoxical findings were resolved by using a “de novo” motif-discovery approach showing that the SXY consensus sequence occupied by NLRC5 in vivo diverges significantly from that occupied by CIITA. These sequence differences were sufficient to determine preferential occupation and transactivation by NLRC5 or CIITA, respectively, and the S box was found to be the essential feature conferring NLRC5 specificity. These results broaden our knowledge on the transcriptional activities of NLRC5 and CIITA, revealing their dependence on shared enhanceosome factors but their recruitment to distinct enhancer motifs in vivo. Furthermore, we demonstrated selectivity of NLRC5 for genes encoding MHCI or related proteins, rendering it an attractive target for therapeutic intervention. NLRC5 and CIITA thus emerge as paradigms for a novel class of transcriptional regulators dedicated for transactivating extremely few, phylogenetically related genes. Major histocompatibility complex class I (MHCI) molecules are central to immunity and immunological disorders, and constitute a major obstacle in organ transplantation. It is therefore vital to gain insight into the regulation of their expression. NLRC5 was recently found to regulate MHCI gene transcription. However, we lack a thorough understanding of its target gene specificity and mechanism of action. Our work addresses these questions, delineating the unique consensus sequence required for NLRC5 recruitment and pinpointing conserved features conferring its specificity. Furthermore, through genome-wide analyses, we confirm that NLRC5 regulates classical MHCI genes and identify novel target genes, all encoding non-classical MHCI molecules exerting an array of functions in immunity and tolerance. We thereby demonstrate that NLRC5 exclusively transactivates genes of the MHCI pathway, rendering it an attractive target for future therapeutic intervention. The most striking feature of NLRC5 is its restricted and highly focused transcriptional activity, which has been described so far only for one related factor, CIITA. NLRC5 and CIITA therefore emerge as prototypes for a novel kind of extremely specific transcriptional regulator.

SERPINA3 (Serpin peptidase inhibitor clade A member 3), also known as a1-antichymotrypsin, is a serine protease inhibitor involved in a wide range of biological processes. Recently, it has been shown to be up-regulated in human placental diseases in association with a hypomethylation of the 5′ region of the gene. In the present study, we show that the promoter of SERPINA3 is transcriptionally activated by three transcription factors (TFs) (SP1, MZF1 and ZBTB7B), the level of induction being dependent on the rs1884082 single nucleotide polymorphism (SNP) located inside the promoter, the T allele being consistently induced to a higher level than the G, with or without added TFs. When the promoter was methylated, the response to ZBTB7B was allele specific (the G allele was strongly induced, while the T allele was strongly down-regulated). We propose an adaptive model to explain the interest of such a regulation for placental function and homeostasis. Overexpression of SERPINA3 in JEG-3 cells, a trophoblast cell model, decreased cell adhesion to the extracellular matrix and to neighboring cells, but protects them from apoptosis, suggesting a way by which this factor could be deleterious at high doses. In addition, we show in different human populations that the T allele appears to predispose to Intra Uterine Growth Restriction (IUGR), while a G allele at a second SNP located in the second exon (rs4634) increases the risk of preeclampsia. Our results provide mechanistic views inside the involvement of SERPINA3 in placental diseases, through its regulation by a combination of epigenetic, genetic and TF-mediated regulations.

Mutations in STOX1 were proposed to be causal for predisposing to preeclampsia, a hypertensive disorder originating from placental defects, affecting up to 10% of human pregnancies. However, after the first study published in 2005 three other groups have dismissed the polymorphism described in the first paper as a causal mutation. In the present study, we have produced a choriocarcinoma cell line overexpressing STOX1. This overexpression results in transcriptional modification of 12.5% of the genes, some of them being direct targets as shown by chromatin immunoprecipitation. STOX1 overexpression correlates strongly and specifically with transcriptomic alterations in preeclamptic placentas (r = 0.30, p = 9.10⁻⁷). Numerous known key modulators of preeclampsia (such as Endoglin, Syncytin, human chorionic gonadotrophin -hCG-, and Glial Cell Missing Homolog -GCM1-) were modified in these transformed choriocarcinoma cells. Our results contribute to reconcile contradictory data concerning the involvement of STOX1 in preeclampsia. In addition, they strongly suggest that anomalies in STOX1 expression are associated with the onset of preeclampsia, thus indicating that this gene should be the target of future studies. Our cellular model could constitute an invaluable resource for studying specific aspects of this human disease.