The molecular events that mediate Ig class switching, a process critical for generating a functionally diverse humoral immune response, are poorly understood. A role for NF-kB/Rel transcription factors in switching is suggested by multiple binding sites for these proteins within the Ig heavy chain locus, including promoters, enhances, and switch regions. Thus, NF-kB/Rel proteins might regulate transcriptional events that determine the accessibility of constant heavy (CH) genes for switching, as well as the switching process itself. Indeed, B cells from mice made genetically deficient in p50/NFkB (p50-/-), the transactivation domain of c-Rel (dc-Rel), or RelA/p65 show distinct defects in germline CH RNA expression and/or switch recombination itself. This proposal will determine the 1) functional roles, 2) expression patterns, and 3) mechanism(s) of action of NFkB/Rel proteins in Ig class switching in normal murine B cells in vitro in response to distinct combinations of activators and cytokines. The ability of NF-kB/Rel proteins to regulate Ig isotype selection through modulation of an enhancer complex 3' to Cha (3'aE) and/or I exon promoters, as well as through changes in the cell cycle will be determined. The functions and expression patterns of NF-kB/Rel knockout (KO) and KO/replacement mice activated with anti-lg-dextran, CD40-ligand, and/or LPS +/- multiple cytokines. The locus of action of NF-kB/Rel proteins in germline CH gene expression will be determined using B cells from 3'aE and/or IXSXCX transgenic mice. We will determine the relationships between 1) Ig class switching by flow cytometry and digestion-circularization PCR assays, 2) germline CH RNA levels by RT-PCR, 3) expression of NF-kB/Rel proteins by EMSA immunoblotting, and in vivo footprinting, 4) transgene activation, and 5) DNA synthesis, cell cycle progression, and apoptosis; CFSE labeling will relate division number with Ig isotype expression. These studies will elucidate key parameters that underlie the molecular basis of Ig switching.
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