Molecular Mechanisms of the Pre-TCR Alpha Chain (pTa) Enhancer Transcriptional Regulation by Notch1.

Abstract 3649

Poster Board III-585

The Notch1 pathway and pre-TCR signalling are critical regulators of thymocyte development that have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Although the pre-TCR is required for Notch-dependent T-ALL, the role of pre-TCR alpha (pTa) as a Notch1 target remains controversial. Previous work in our laboratory has shown that the pTa gene is a target gene of the E2A-HEB transcription factors and that the SCL and LMO1 or LMO2 oncogenic transcription factors inhibits E2A activity and pTa expression, thereby causing thymocyte differentiation arrest during the pre-leukemic stage. SCL and LMO1/2 are implicated in 25% of T-ALL cases and induce aggressive T-ALL in transgenic (tg) mice when inappropriately expressed in the thymus. To define the mechanism regulating pTa expression by Notch1 and SCL, we used pTa regulatory sequences as a tool. To this end, we have performed transcriptional assays by co-delivering a luciferase reporter gene construct driven by the 200 bp pTa enhancer sequence with or without SCL or Notch1 intracellular domain (N1ICD), the functional fragment of the Notch1 receptor. Sequence analysis of the pTa enhancer revealed the presence of four E boxes that are potential binding sites for E2A-HEB and a CSL consensus sequence that can recruit the CSL/RBP-Jk transcription factor. Transcriptional assays in double negative thymomas indicated that the integrity of E box 2 and 3 (that recruit E2A-HEB) are essential for pTa enhancer activity in DN thymocytes, confirming the importance of E2A-HEB for pTa expression in these cells. In contrast, the CSL consensus sequence is important but not essential for pTa enhancer activity in these cells, consistent with previous results based on a conditional Notch1 knock out. In the presence of N1ICD, pTa enhancer sequences are strongly activated, either in fibroblasts or in double negative T cell lines in a dose dependent manner. This activation requires the integrity of the CSL consensus sequence but not of the E boxes. Moreover, we found that N1ICD strongly overrides the inhibitory activity of SCL on the pTa enhancer. Inhibition by SCL requires the integrity of E2 and E3 but not the CSL, confirming that SCL inhibits E2A-HEB activity, without affecting N1ICD. Together, our observations indicate that the regulation of pTa enhancer activity by Notch1 and E2A/HEB occur though independent mechanisms.