COOPERATIVE INTERACTION BETWEEN THE DNA-BINDING DOMAINS OF PU.1 AND IRF4

The two lymphoid-specific transcription factors PU.1 and IRF4 form a c ooperative ternary complex at immunoglobulin enhancer elements such as the lambda B and kappa E3' sites. We report here that the synergy of this interaction can be reconstituted in part with the DNA-binding dom ains of the two proteins The minimal DNA binding-domain of IRF4 was ma pped to residues 20 to 137, corresponding to the conserved DNA-binding region of other interferon regulatory factors (IRFs). This domain can bind weakly to a synthetic murine lambda B element, while IRF4 constr ucts that contain residues 1 to 19 require the presence of PU.1 for DN A-binding at similar concentrations. Fluorescence polarization of fluo rescein-labelled DNA was used to show that the presence of residues 1 to 19 decreases the binding affinity of IRF4 N-terminal constructs fro m two- to fivefold. However, all constructs bound better to the lambda B element in the presence of the DNA-binding domain of PU.1. This coo perative interaction was not dependent on phosphorylation of the PEST domain of PU.1, but was dependent on the proper spacing of the binding sites for PU.I and IRF4. These data suggest that at least part of the cooperative interaction between full-length PU.1 and IRF4 involves th e DNA-binding domains of the two proteins. NMR spectroscopy of N-15-la belled PU.1 and IRF4 constructs indicates that the PEST domain of PU.1 and residues 1 to 19 of IRF4 may be unstructured in the isolated prot eins. (C) 1998 Academic Press.