Notch1 augments NF-κB activity by facilitating its nuclear retention

Notch1 specifically upregulates expression of the cytokine interferon-γ in peripheral T cells through activation of NF-κB. However, how Notch mediates NF-κB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF-κB induction during T-cell activation. NF-κB activation occurs within minutes of T-cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used γ-secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF-κB activation, but did not affect the initial activation of NF-κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1^IC) directly interacts with NF-κB and competes with IκBα, leading to retention of NF-κB in the nucleus. Additionally, we show that N1^IC can directly regulate IFN-γ expression through complexes formed on the IFN-γ promoter. Taken together, these data suggest that there are two 'waves' of NF-κB activation: an initial, Notch-independent phase, and a later, sustained activation of NF-κB, which is Notch dependent.