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

T-cell differentiation requires Notch1 signaling. Here we show that an enhancer upstream of Notch1 active in double-negative (DN) 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-lineage commitment and TCR? V-DJ recombination, Cd3 expression and ?-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: 34 bulk RNAseq, 4 single-cell RNAseq