E2A and HEB activate the pre-TCR alpha promoter during immature T cell development

The pre-TCR alpha (pT alpha) is exclusively expressed in immature thymocyte s and constitutes the pre-TCR complex with TCR beta, which regulates early T cell differentiation. Despite the recent identification of the pT alpha e nhancer, the contribution of the promoter region, the direct DNA-protein in teraction, and the regulation of such interaction along with T cell develop ment have not been investigated. We analyzed the pT alpha promoter region a nd identified the critical elements for transcription of the pT alpha gene. The pT alpha promoter was found to contain two consecutive E-box elements that are critical for pT alpha transcription. The E-box elements in the pro moter region formed the specific DNA-protein complex that was exclusively o bserved in immature thymocytes, not in mature thymocytes and T cells. The E proteins in this complex were identified as E2A and HeLa E-box binding pro tein (HEB), and overexpression of E2A and HEB resulted in activation of the pT alpha promoter. The binding complex in the consecutive E-boxes in the p Ta promoter changed along with T cell development, as a distinct DNA-bindin g complex was observed in mature T cells. Comparing the E-box regions in th e enhancer and the promoter, those in the promoter appear to make a greater contribution to pT alpha gene transcription.