TRANSCRIPTION FACTOR PIP CAN ENHANCE DNA-BINDING BY E47, LEADING TO TRANSCRIPTIONAL SYNERGY INVOLVING MULTIPLE PROTEIN DOMAINS

The transcription factors E2A (E12/E47) and Pip are both required for normal E-cell development. Each protein binds to regulatory sequences within various immunoglobulin enhancer elements. Activity of E2A prote ins can be regulated by interactions with other proteins which influen ce their DNA binding or activation potential. Similarly, Pip function can be influenced by interaction with the protein PU.1, which can recr uit Pip to bind to DNA. We show here that a previously unidentified Pi p binding site resides adjacent to the E2A binding site within the imm unoglobulin kappa 3' enhancer. Both of these binding sites are crucial for high-level enhancer activity. We found that E47 and Pip can funct ionally interact to generate a very potent 100-fold transcriptional sy nergy. Through a series of mutagenesis experiments, we identified the Pip sequences necessary for transcriptional activation and for synergy with E47. Two synergy domains (residues 140 to 207 and 300 to 420) in addition to the Pip DNA binding domain (residues 1 to 134) are requir ed for maximal synergy with E47. We also identified a Pip domain (resi dues 207 to 300) that appears to mask Pip transactivation potential. P art of the synergy mechanism between E47 and Pip appears to involve th e ability of Pip to increase DNA binding by E47, perhaps by inducing a conformational change in the E47 protein. E47 may also induce a confo rmational change in Pip which unmasks sequences important for transcri ptional activity. Based upon our results, we propose a model for E47-P ip transcriptional synergy.