Functional synergism of STAT6 with either NF-kappa B or PU.1 to mediate IL-4-induced activation of IgE germline gene transcription

Ig heavy chain class switching to IgE is directed by IL-4 and IL-13 by indu cing transcription from the IgE germline promoter. a crucial transcription factor in this process is STAT6, which binds to a specific DNA element upon cytokine activation. In this paper it is shown that the B cell- and monocy te-specific factor PU.1 interacts,vith a closely spaced sequence in the hum an IgE germline promoter that overlaps with a previously described binding site for NF kappa B/rel, The authenticity of PU.1 was demonstrated by speci fic competition and supershifts in EMSA experiments. In addition, in vitro translated PU.1 could interact with an oligonucleotide derived from the IgE germline promoter containing the PU.1 binding site and migrated with the s ame mobility compared with the complex formed with nuclear extracts, Transi ent transfection experiments using IgE germline promoter reporter gene cons tructs demonstrated that mutations affecting DNA binding of PU.1 or NF kapp a B/rel had no or little effect on IL-4 inducibility of these plasmids. How ever, point mutations that abolished binding of both factors abrogated cyto kine inducibility. No strict spacing of the STAT6 and the composite PU.1/NF -kappa B elements is required for IL-4 induction, IL-4-induced STAT6 DNA bi nding was retained in PU.1(-)/NF kappa B/rel(-) double mutants. The data de monstrate that cooperation of STAT6 with at least PU.1 or NF kappa B/rel is necessary for IL-4-induced activation of IgE germline gene transcription.