COACTIVATOR OBF-1 MAKES SELECTIVE CONTACTS WITH BOTH THE POU-SPECIFICDOMAIN AND THE POU HOMEODOMAIN AND ACTS AS A MOLECULAR CLAMP ON DNA

The lymphoid-specific transcriptional coactivator OBF-1 (also known as OCA-B or Bob-1) is recruited to octamer site-containing promoters by interacting with Oct-1 or Oct-2 and thereby enhances the transactivati on potential of these two Oct factors, For this interaction the POU do main is sufficient. By contrast, OBF-1 does not interact with the POU domains of other POU proteins, such as Oct-4, Oct-6, or Pit-1, even th ough these factors bind efficiently to the octamer motif, Here we exam ined the structural requirements for selective interaction between the POU domain and OBF-1, Previous data have shown that formation of a te rnary complex among OBF-1, the POU domain, and the DNA is critically d ependent on residues within the octamer site, By methylation interfere nce analysis we identified bases that react differently in the presenc e of OBF-1 compared to the POU domain alone, and using phosphothioate backbone-modified probes in electrophoretic mobility shift assays, we identified several positions influencing ternary complex formation, We then used Oct-1/Pit-1 POU domain chimeras to analyze the selectivity of the interaction between OBF-1 and the POU domain, This analysis ind icated that both the POU specific domain (POU,) and the POU homeodomai n (POU,) contribute to complex formation. Amino acids that are differe nt in the Pit-1 and Oct-1 POU domains and are considered to be solvent accessible based on the Oct-1 POU domain/DNA cocrystal structure were replaced,vith alanine residues and analyzed for their influence on co mplex formation, Thereby, we identified residues L6 and E7 in the POUs and residues K155 and 1159 in the POUH to be critical in vitro and in vivo for selective interaction with OBF-1, Furthermore, in an in vivo assay we could show that OBF-1 is able to functionally recruit two ar tificially separated halves of the POU domain to the promoter DNA, the reby leading to transactivation, These data allow us to propose a mode l of the interaction between OBF-1 and the POU domain, whereby OBF-1 a cts as a molecular clamp holding together the two moieties of the POU domain and the DNA.