Identification of a stimulus-dependent DNase I hypersensitive site betweenthe I alpha and C alpha exons during immunoglobulin heavy chain class switch recombination

The complete humoral response to foreign antigen depends upon tno distinct recombination events within the heavy chain locus of immunoglobulin. The Fi rst recombination event takes place in what will become the antigen combini ng site of the antibody molecule, encoded by V, D and J segments. The secon d recombination event involves the looping-out of large spans of DNA which separate the various dusters of heavy chain exons which define the differen t immunoglobulin isotypes. or classes, While a great deal has been learned about the nature of the VDJ recombinase, very little is known about the nat ure of the class-switch recombinase, Using a cell, system where class-switc h recombination occurs primarily to the IgA locus, ne have looked for stimu lus-dependent changes in the chromatin structure of the IgA locus which mig ht result from interactions between components of the recombinase and cis-e lements within the region. We present evidence that strongly suggests that the class-switch recombinase interacts between the I alpha and C alpha exon s of IgA, just upstream of the highly reiterated DR1 and DR2 elements, Howe ver, although multiple potential SMAD-4 sites are located precisely within the DNase I hypersensitive site and 160 bp upstream of that site, ne failed to detect any evidence of DNA/protein interactions near the hypersensitive site, Moreover, recombinant SMAD-3/4 proteins fail to interact with these sites with appreciable affinity in vitro. These data suggest that same othe r structural alteration at this site (e.g. RNA/DNA hybrid) mag mediate the nuclease sensitivity. (C) 2000 Federation of European Biochemical Societies .