MOLECULAR BASIS OF IMMUNOGLOBULIN HEAVY CHAIN SWITCH

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When mature B lymphocytes, which express IgM and IgD on their surface, are activated by antigen and receive accessory signals, they switch to express downstream heavy chain constant region (CH) genes while maintaining the same expressed variable region domain. This class switch changes the ability of the antibody to clear various pathogens from various sites in the body since the CH region determines the effector function of the antibody. Antibody class switching is effected by a DNA recombination event called switch recombination, which occurs within or near 2 to 10 kb switch (S) regions containing tandemly repeated sequences located 5' of each CH gene, except Cdelta. The means by which the DNA is cut, aligned and rejoined is unknown, as are the roles of several nuclear proteins that have been shown to bind to S regions. it has become clear that switching is directed by induction of accessibility of particular CH genes prior to class switch recombination since the unrearranged, or germline, CH genes to which a B cell will subsequently undergo class switching are transcribed prior to switching to that particular CH gene. Furthermore, this transcription is induced by cytokines that direct switching to particular CH genes and promoters for these germline CH transcripts are regulated by cytokines. This grant is a proposal to continue studies that have been funded for 9 years on the mechanism and regulation of antibody class switching, concentrating on the switch to IgA, using a mouse cell line l.29mu that undergoes this process in culture. Mouse splenic B cells will be used to verify the results with I.29mu cells, when feasible. Gene targeting will be used to determine the function of germline transcription, asking whether the RNA itself or protein binding sites 5' to tandem repeated switch sequences are required in addition to transcription. Experiments to create transfectable plasmids that can undergo class switching will be continued in order to define DNA sequences required for switching and to assay the function of genes that affect class switching. Effort will be directed toward the characterization and cloning of the gene encoding a protein(s) that binds to novel TGFbeta-response elements in the promoter for germline Calpha transcripts that were identified since the last competitive renewal. Genes encoding mRNAs that are up or downregulated in cells induced to undergo class switching will be cloned and characterized. During the previous cycle this group has shown that inhibitors of the nuclear enzyme which aids in DNA repair, poly (ADP-ribose) polymerase increase switch recombination and efforts will now be directed toward understanding the mechanism of this stimulation. The sites and kinetics of induction of the endonuclease cutting events that must be required to initiate switch recombination will be examined.