Cooperation among multiple transcription factors is required for access to minimal T-cell receptor α-enhancer chromatin in vivo

To understand the molecular basis for the dramatic functional synergy between transcription factors that bind to the minimal T-cell receptor alpha enhancer (E alpha), we analyzed enhancer occupancy in thymocytes of transgenic mice in vivo by genomic footprinting. We found that the formation of a multiprotein complex on this enhancer in vivo results from the occupancy of previously identified sites for CREB/ATF, TCF/LEF, CBF/PEBP2, and Ets factors as well as from the occupancy of two new sites 5' of the CRE site, GC-I (which binds Sp1 in vitro) and GC-II. Significantly, although all sites are occupied on a wild-type E alpha, all sites are unoccupied on versions of E alpha with mutations in the TCF/LEF or Ets sites. Previous in vitro experiments demonstrated hierarchical enhancer occupancy with independent binding of LEF-1 and CREB, Our data indicate that the formation of a multiprotein complex on the enhancer in vivo is highly cooperative and that no single E alpha binding factor can access chromatin in vivo to play a unique initiating role in its assembly. Rather, the simultaneous availability of multiple enhancer binding proteins is required for chromatin disruption and stable bidding site occupancy as well as the activation of transcription and V(D)J recombination.