E2A and HEB Activate the Pre-TCRα Promoter During Immature T Cell Development

Abstract

The pre-TCRα (pTα) is exclusively expressed in immature thymocytes and constitutes the pre-TCR complex with TCRβ, which regulates early T cell differentiation. Despite the recent identification of the pTα enhancer, the contribution of the promoter region, the direct DNA-protein interaction, and the regulation of such interaction along with T cell development have not been investigated. We analyzed the pTα promoter region and identified the critical elements for transcription of the pTα gene. The pTα promoter was found to contain two consecutive E-box elements that are critical for pTα transcription. The E-box elements in the promoter region formed the specific DNA-protein complex that was exclusively observed 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 protein (HEB), and overexpression of E2A and HEB resulted in activation of the pTα promoter. The binding complex in the consecutive E-boxes in the pTα promoter changed along with T cell development, as a distinct DNA-binding complex was observed in mature T cells. Comparing the E-box regions in the enhancer and the promoter, those in the promoter appear to make a greater contribution to pTα gene transcription.

Human T-Cell Leukemia Virus Type 1 Tax Protein Down-Regulates Pre-T-Cell Receptor Alpha Gene Transcription in Human Immature Thymocytes

“…In cotransfection experiments performed with EP180⌬E (from which the two E-boxes in the promoter region were deleted) or EmP180 (in which base substitutions were introduced in the three E-box sites in the enhancer region), luciferase activity could not be detected in the presence of E47. These observations confirm previous results demonstrating that E-box elements in both the enhancer and promoter regions are regulating the activation of the pT␣ gene (30,40).…”

“…Thus, the overexpression of E47, one of the two E2A proteins, and its binding to E-box sequences present in the promoter of the pT␣ gene resulted in increased transcription of this gene (27,30,40). In the present study, we first showed in transienttransfection experiments that the HTLV-1 Tax protein downregulated the activation of the pT␣ promoter mediated by the ectopic expression of E47.…”

“…The pT␣ reporter element EP180, together with the two mutants EP180 ⌬E and EmP180 containing deletion or base substitutions of the E-boxes, respectively, were a generous gift from A. Takeuchi (40). The Tax expression plasmid, pCMV-Tax, containing the tax coding sequence under the control of the cytomegalovirus promoter, was a generous gift from W. C. Greene.…”

“…When assembled at the cell membrane, the pre-TCR complex transmits an essential survival signal for the development of T cells and is thought to play roles in allelic exclusion of TCR␤. The transcription of the pT␣ gene has been shown to be regulated through the basic helix-loop-helix (bHLH) transcription factors, also called E proteins (27), which bind specific sequences (E-box; CANNTG) contained in the regulatory sequences of this gene (30,40). Up-regulation of pT␣ transcription coincides with irreversible T-cell lineage commitment in the thymus and is mainly observed in immature thymocytes (5,6,43).…”

Combined expression of pTα and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis

“…Whether such a regulation occurs at the transcriptional level needs to be elucidated. Indeed, the pT␣ promoter has been recently described to carry E2A-and HEB-responsive E box cis-elements, although their functional characterization still requires full elucidation (23)(24)(25). Nevertheless, the transcriptional function of such bHLH E proteins has been described as inhibited by Notch signaling, either by way of the Ras pathway (26) or by the Notch-target HES-1 (27).…”