Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A

Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo beta-selection, during which cells expressing functionally rearranged TCRbeta proliferate and differentiate into CD4(+)CD8(+) progeny, Notch1 signaling is abruptly down-regulated. In this report, we investigate the mechanisms that control Notch1 expression during thymopoiesis. We show that Notch1 and E2A directly regulate Notch1 transcription in pre-beta-selected thymocytes. Following successful beta-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-beta-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. Our findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions.