NF-kappa B p50-dependent in vivo footprints at Ig S gamma 3 DNA are correlated with mu-->gamma 3 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.
Wuerffel
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Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60680, USA.
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Kenter
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D000818:Animals; D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D018983:DNA Footprinting / Q000379:methods; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology; D005854:Germ Cells / Q000276:immunology; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D007142:Immunoglobulin gamma-Chains / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007148:Immunoglobulin mu-Chains / Q000235:genetics* / Q000378:metabolism; D007399:Interphase / Q000276:immunology; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D008819:Mice, Nude; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D052002:NF-kappa B p50 Subunit; D009698:Nucleoproteins / Q000378:metabolism; D011995:Recombination, Genetic / Q000276:immunology*; D014158:Transcription, Genetic / Q000276:immunology
D004268:DNA-Binding Proteins; D007142:Immunoglobulin gamma-Chains; D007148:Immunoglobulin mu-Chains; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009698:Nucleoproteins