NF-kappa B p50-dependent in vivo footprints at Ig S gamma 3 DNA are correlated with mu ->gamma 3 switch recombination

NF-kappaB has been demonstrated to play critical roles in multiple aspects of immune responses including Ig H chain isotype switching. To better defin e the specific roles the p50 subunit of NF-kappaB plays in mu-->gamma3 swit ch recombination (SR), we systematically evaluated p50-deficient B cells fo r activities that are strongly correlated with SR. B cell activation with L PS plus anti-IgD-dextran plus IL-5 plus IL-4 plus TGF-beta produced normal levels of proliferation and gamma3 germline transcripts in p50-deficient B cells, but mu-->gamma3 SR was impaired. In vitro binding studies previously showed that NF-kappaB p50 homodimer binds the switch nuclear B-site protei n (SNIP) of the S gamma3 tandem repeat. Ligation-mediated PIER in vivo foot print analysis demonstrates that the region spanning the SNIP and switch nu clear A-site protein (SNAP) binding sites of the S gamma3 region are contac ted by protein in normal resting splenic B cells. B cells that are homozygo us for the targeted disruption of the gene encoding p50 (-/-) show strong a berrant footprints, whereas heterozygous cells (+/-) reveal a partial effec t in S gamma3 DNA. These studies provide evidence of nucleoprotein interact ions at switch DNA in vivo and suggest a direct interaction of p50 with S g amma3 DNA that is strongly correlated with SR competence.