TET proteins regulate Drosha expression and impact microRNAs in iNKT cells

DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As Drosha is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression. To assess the impact of TET2 and TET3 loss in small RNA biogenesis, we isolated RNA from wild type and Tet2/3 DKO thymic iNKT cells and we performed small-RNA sequencing to compare small RNA expression.DNA去甲基化酶TET2与TET3通过介导谱系特异性因子编码基因的DNA去甲基化，在胸腺恒定自然杀伤T（iNKT）细胞分化过程中发挥核心作用。矛盾的是，差异基因表达分析显示，在iNKT细胞中敲除TET2和TET3后，大量基因出现上调表达。若TET蛋白缺失可降低基因表达抑制因子的表达水平，则可解释这一意外发现。本研究发现，TET2与TET3可通过在基因本体区域生成5-羟甲基胞嘧啶（5hmC）并影响染色质可及性，协同调控Drosha的表达。鉴于Drosha参与微小RNA（microRNA）的生物发生过程，本研究进一步探究了TET2/3缺失对iNKT细胞内微小RNA表达的影响。本研究发现，在表达下调的微小RNA中包含Let-7家族成员，该家族可在体内抑制iNKT细胞谱系特异性因子早幼粒细胞白血病锌指蛋白（PLZF）的表达。本研究数据将TET蛋白与微小RNA表达联系起来，揭示了TET蛋白介导基因表达调控的另一层机制。为评估TET2与TET3缺失对小RNA生物发生的影响，本研究从野生型及Tet2/3双敲除（DKO）胸腺iNKT细胞中提取RNA，并通过小RNA测序（small-RNA sequencing）比较两组的小RNA表达差异。