The long-term objective of this proposal is to elucidate the mechanisms underlying helper T (Th) cell-mediated regulation and targeting of isotype switching in murine B-cells. Cytokines such as IL-4 are believed to target switch recombination to specific isotypes by inducing germline Ig gene transcription, possibly making the switch regions accessible to a putative switch recombinase. T-cell contact-dependent signals required for isotype switching are provided by the CD40 ligand (CD40L)-CD40 interaction, but nothing is known about how these signals regulate isotype switching. The investigation has shown that recombinant CD40L induces expression of the germline gamma-1 and epsilon Ig genes in an IL-4 independent manner, and that IL-4 and CD40L synergize to promote maximal germline transcript expression. These results and results from other laboratories suggest that signals delivered via the CD40L-CD40 interaction may regulate isotype switching, at least in part, at the level of germline Ig gene transcription. The central goals of this proposal are to characterize in detail the mechanisms underlying CD40-mediated regulation of germline gamma-1 Ig gene transcription and to determine the role of CD40-mediated signals in activating the gamma-1 switch region for recombination in vivo. A PCR-based assay will be used to measure switch recombination of Sgamma-1 and to determine if and when CD40L and cytokines are required for activating the DNA recombination event. The effects of CD40-mediated signals on germline gamma-1 transcript expression will be characterized in detail by RNAase protection, nuclear run-on transcription assays and mRNA stability studies. CD40-responsive, cis-acting elements will be identified and mapped by transient transfection of germline gamma-1 promoter-luciferase reporter gene constructs. CD40-responsive DNA-binding proteins responsible for regulation of germline gamma-1 Ig gene transcription will be identified by EMSA, binding sites will be defined by DNA footprinting, and function assessed by site-specific mutagenesis and transfection of germline gamma-1 promoter-reporter gene constructs. Novel DNA-binding proteins will be isolated biochemically and/or by cDNA cloning and characterized in detail. Finally, the in vivo role of CD40-responsive germline gamma-1 promoter-binding proteins in regulating endogenous germline gamma-1 transcription and switch recombination to Sgamma-1 will be determined using a combination of protein and gene knock-out strategies.
NIH ExPorter
NIH ExPorter