Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, Germany; Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.
Cell Immunol. 2022 Jan;371:104471. doi: 10.1016/j.cellimm.2021.104471. Epub 2021 Dec 20.
Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.
FOXP3-TSDR（调节性 T 细胞特异性去甲基化区域）的去甲基化是调节性 T（Treg）细胞稳定分化和抑制功能的标志。以前旨在实现人类初始 T 细胞分化的方案未能实现 Treg 细胞特异性的表观遗传特征。Ten-eleven translocation（TET）酶催化 DNA 去甲基化。构建了包含无功能 Cas9、TET1 的催化结构域、蓝色荧光蛋白以及靶向 FOXP3-TSDR 特定片段的单指导 RNA（sgRNA）的融合蛋白表达质粒，并转染 Jurkat T 细胞。通过深度扩增子亚硫酸氢盐测序分析 FOXP3-TSDR 甲基化，通过实时 PCR 测定细胞 Foxp3、Tbet、Gata3 和 Rorgt mRNA 水平。dCas9TET1 的过表达显著降低了 Jurkat 细胞 FOXP3-TSDR 的甲基化水平，并增加了 Foxp3 mRNA 的表达，而其他主要 T 细胞谱系的主转录因子 mRNA 的表达基本不受影响。dCas9-TET1 构建体转染介导患者原代 T 细胞的 Treg 编程可能是可行的。
