C-MYB AND CORE-BINDING FACTOR PEBP2 DISPLAY FUNCTIONAL SYNERGY BUT BIND INDEPENDENTLY TO ADJACENT SITES IN THE T-CELL RECEPTOR DELTA-ENHANCER

A T-cell-specific transcriptional enhancer lies within the J(delta)3-C (delta)intron of the human T-cell receptor delta gene, We have previou sly shown that a 30-bp element, denoted delta E3, acts as the minimal TCR delta enhancer and that within delta E3, adjacent and precisely sp aced binding sites for core-binding factor (CBF/PEBP2) and c-Myb are e ssential for transcriptional activity. These data suggested that CBF/P EBP2 and c-Myb synergize to mediate transcriptional activity but did n ot establish the molecular basis for synergy. In this study, we have e xamined in detail the binding of CBF/PEBP2 and c-Myb to delta E3, We f ound that CBF/PEBP2 and c-Myb could simultaneously occupy the core sit e and one of two overlapping Myb sites within delta E3. However, equil ibrium binding and kinetic dissociation experiments suggest that the t wo factors bind to delta E3 independently, rather than cooperatively, This was found to be true by using isoforms of these factors present i n extracts of transfected COS-7 cells, as well as the natural factors present in nuclear extracts of the Jurkat T-cell line, We further show ed that CBF/PEBP2 and c-Myb provide unique transactivation functions, since the core-Myb combination cannot be substituted by dimerized core or Myb sites, We propose that spatially precise synergy between CBF/P EBP2 and c-Myb may result from the ability of the two factors to form a composite surface that makes unique and stereospecific contacts with one or more additional components of the transcriptional machinery.