Distinct requirement of Runx complexes for TCRβ enhancer activation at distinct developmental stages

A TCR beta enhancer, known as the E beta enhancer, plays a critical role in V(D)J recombination and transcription of the Tcrb gene. However, the coordinated action of trans-acting factors in the activation of E beta during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the E beta function. A single mutation at one of the two Runx binding motifs within the E beta severely impaired Tcrb activation at the initiation phase in immature thymocytes. However, TCR beta expression level in mature thymocytes that developed under such a single Runx site mutation was similar to that of the control. In contrast, mutations at two Runx motifs eliminated E beta activity, demonstrating that Runx complex binding is essential to initiate E beta activation. In cells expressing Tcrb harboring rearranged V(D)J structure, Runx complexes are dispensable to maintain TCR beta expression, whereas E beta itself is continuously required for TCR beta expression. These findings imply that Runx complexes are essential for E beta activation at the initiation phase, but are not necessary for maintaining E beta activity at later developmental stages. Collectively, our results indicate that the requirements of trans-acting factor for E beta activity are differentially regulated, depending on the developmental stage and cellular activation status.