A double negative thymocyte specific enhancer augments Notch1 signaling to direct early T cell progenitor expansion, lineage restriction and -selection.. A double negative thymocyte specific enhancer augments Notch1 signaling to direct early T cell progenitor expansion, lineage restriction and -selection.

T cell differentiation requires Notch1 signaling. Here we show that an enhancer upstream of Notch1 active in double-negative (DN) mouse thymocytes is responsible for raising Notch1 signaling intra-thymically. This enhancer is required to expand multipotent progenitors intra-thymically while delaying early differentiation until lineage restrictions are established. Early thymic progenitors lacking the enhancer show accelerated differentiation through the DN stages and increased frequency of B-, ILC-, and NK-cell differentiation. Transcription regulators for T cell lineage restriction and commitment are expressed normally, but ILC- and NK-cell gene expression persists after T cell lineage commitment and TCRb V-DJ recombination, Cd3 expression and b-selection are impaired. This Notch1 enhancer is inactive in double-positive (DP) thymocytes. Its aberrant reactivation at this stage in Ikaros mutants is required for leukemogenesis. Thus, the DN-specific Notch1 enhancer harnesses the regulatory architecture of DN and DP thymocytes to achieve carefully orchestrated changes in Notch1 signaling required for early lineage restrictions and normal T cell differentiation. Overall design: 3 ChIP-seq samples

T细胞分化依赖于Notch1信号通路。本研究证实，小鼠双阴性（double-negative, DN）胸腺细胞中具有活性的Notch1基因上游增强子，负责在胸腺内提升Notch1信号通路的活性水平。该增强子对于胸腺内扩增多能祖细胞、并将早期分化进程阻滞至谱系限制性建立完成前，是必需的。缺失该增强子的早期胸腺祖细胞，其DN阶段的分化进程会加速，且B细胞、固有淋巴细胞（innate lymphoid cell, ILC）以及自然杀伤（natural killer, NK）细胞的分化频率显著升高。尽管T细胞谱系限制性与定型相关的转录调控因子表达正常，但在T细胞谱系定型以及T细胞受体β链（TCRβ）V-DJ重排完成后，ILC与NK细胞相关基因的表达仍持续存在；同时Cd3分子表达与β选择过程均受到损伤。该Notch1增强子在双阳性（double-positive, DP）胸腺细胞中无转录活性。在伊卡洛斯（Ikaros）突变体中，该增强子在此发育阶段的异常重激活，对于白血病发生过程是必需的。综上，DN特异性Notch1增强子通过调控双阴性与双阳性胸腺细胞的基因表达网络，精准调控Notch1信号通路的动态变化，以满足早期谱系限制性建立与正常T细胞分化的需求。实验整体设计：共3组染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）样本。