NF-κB p50-Dependent In Vivo Footprints at Ig Sγ3 DNA Are Correlated with μ→γ3 Switch Recombination

NF-kappa B has been demonstrated to play critical roles in multiple aspects of immune responses including Ig H chain isotype switching. To better define the specific roles the p50 subunit of NF-kappa B plays in mu-->gamma 3 switch recombination (SR), we systematically evaluated p50-deficient B cells for activities that are strongly correlated with SR. B cell activation with LPS 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-->gamma 3 SR was impaired. In vitro binding studies previously showed that NF-kappa B p50 homodimer binds the switch nuclear B-site protein (SNIP) of the S gamma 3 tandem repeat. Ligation-mediated PCR in vivo footprint analysis demonstrates that the region spanning the SNIP and switch nuclear A-site protein (SNAP) binding sites of the S gamma 3 region are contacted by protein in normal resting splenic B cells. B cells that are homozygous for the targeted disruption of the gene encoding p50 (-/-) show strong aberrant footprints, whereas heterozygous cells (+/-) reveal a partial effect in S gamma 3 DNA. These studies provide evidence of nucleoprotein interactions at switch DNA in vivo and suggest a direct interaction of p50 with S gamma 3 DNA that is strongly correlated with SR competence.