ELF3 activated by a super-enhancer and an autoregulatory feedback loop is required for high level HLA-C expression on extravillous trophoblasts. ELF3 activated by a super-enhancer and an autoregulatory feedback loop is required for high level HLA-C expression on extravillous trophoblasts

HLA-C arose during evolution of pregnancy in the Great Apes 10-15 million years ago. It has a dual function on placental extravillous trophoblasts (EVT) as it contributes to both tolerance and immunity at the maternal-fetal interface. The mode of its regulation is of considerable interest in connection with the biology of pregnancy and pregnancy abnormalities. First trimester primary EVT in which HLA-C is highly expressed, as well as JEG3, an EVT model cell lines, were employed. Single cell RNA-seq data and quantitative PCR identified high expression of the transcription factor ELF3 in those cells. ChIP-PCR confirmed that both ELF3 and MED1 bound to the proximal HLA-C promoter region. However, binding of RFX5 to this region was absent or severely reduced and the adjacent HLA-B locus remained closed. Expression of HLA-C was inhibited by ELF3 siRNAs and by wrenchnolol treatment. Wrenchnolol is a cell-permeable synthetic organic molecule that mimics ELF3 and is relatively specific for binding to ELF3’s coactivator, MED23, as our data also showed in JEG3. Moreover, the ELF3 gene is regulated by a super-enhancer that spans more than 5 Mb, identified by ATAC-seq, as well as by its sensitivity to (+)-JQ1 (inhibitor of BRD4). ELF3 bound to its own promoter, thus creating an autoregulatory feedback loop that establishes expression of ELF3 and HLA-C in trophoblasts. Wrenchnolol blocked binding of MED23 to ELF3, thus disrupting the positive feedback loop that drives ELF3 expression, with down regulation of HLA-C expression as a consequence. Overall design: To assess the chromatin accessibility of somatic cell lines and trophoblast model cell lines, ATAC-seq was applied to cell line BJ fibroblasts, Hela, JEG3, BeWo and Jar.

人类白细胞抗原C（HLA-C）于1000万至1500万年前的类人猿妊娠演化过程中起源。其在胎盘绒毛外滋养层细胞（extravillous trophoblasts, EVT）上发挥双重功能，可在母胎界面同时参与免疫耐受与免疫调控过程。该分子的调控机制与妊娠生物学及妊娠异常紧密相关，因此受到学界广泛关注。

本研究选用了高表达HLA-C的妊娠早期原代绒毛外滋养层细胞，以及EVT模型细胞系JEG3。通过单细胞RNA测序与定量聚合酶链反应（quantitative PCR）分析，研究人员在上述细胞中检测到转录因子ELF3呈高表达水平。染色质免疫沉淀-聚合酶链反应（ChIP-PCR）实验证实，ELF3与MED1均可结合至HLA-C的近端启动子区域；但调控因子RFX5在此区域的结合则完全缺失或显著降低，且相邻的HLA-B基因座始终处于染色质封闭状态。

ELF3小干扰RNA（small interfering RNAs, siRNAs）与wrenchnolol处理均可抑制HLA-C的表达。Wrenchnolol是一种可穿透细胞膜的合成有机小分子，其结构模拟ELF3，且对ELF3的辅激活因子MED23具有较高结合特异性，本研究在JEG3细胞中也验证了这一特性。此外，通过转座酶可及性测序（ATAC-seq）鉴定发现，ELF3基因受一段跨度超过5 Mb的超级增强子调控，且其表达对BRD4抑制剂(+)-JQ1具有敏感性。ELF3可结合自身启动子，从而形成自身调控反馈环路，维持滋养层细胞中ELF3与HLA-C的表达。

Wrenchnolol可阻断MED23与ELF3的结合，进而破坏驱动ELF3表达的正反馈环路，最终导致HLA-C表达下调。

整体实验设计：为评估体细胞系与滋养层模型细胞系的染色质可及性，本研究对BJ成纤维细胞、海拉（Hela）细胞、JEG3、BeWo及Jar细胞系开展了转座酶可及性测序（ATAC-seq）。