We are studying the immunoglobulin isotype switch induced by interleukins -4, -10 and -13 in B lymphocytes, focusing on the events at the DNA level. To ascertain whether switch factors such as IL-4 act through DNA elements other than the known motifs in promoters upstream of certain immunoglobulin heavy chain constant region genes, we have been trying to locate an expected human homolog of the locus control region (LCR) that has been identified in mouse lying downstream of the C-alpha gene. In mouse this regulatory region has profound effects on isotype switching, as judged from the absence of certain isotypes from the serum of mice in which one component of the LCR has been eliminated by homologous recombination. We have obtained evidence of several DNase I hypersensitive (HS) sites lying downstream of each of the two human C-alpha heavy chain genes; similar sites marked the position of the murine LCR components. To analyze potential regulatory regions corresponding to these sites, we have cloned DNA extending about 50 kb downstream of the C-alpha-2 gene; from the homologous region downstream of C-alpha-1 we have about 30 kb, and we are working to fill in a gap of about 20 kb. So far we have studied luciferase reporter constructs corresponding to one of the DNase I HS sites that is not B cell-specific. This HS site did not have detectable enhancer activity, nor did several flanking DNA segments. Further systematic constructions covering all the DNase I hypersensitivity regions are in progress, as are comparisons of HS sites in human cell lines representative of different stages of B cell maturation. Extensive characterization by sequence analysis of selected regions of the cloned DNA is continuing in an effort to locate regions homologous to the murine LCR. To date this sequence analysis has revealed some features that are interesting from the perspective of the evolutionary development of the immunoglobulin heavy chain locus. We found that each of the two C-alpha genes is flanked by a processed pseudogene derived from the elk-1 gene, which encodes an ETS-related transcription factor known to participate in formation of a ternary complex at the c-fos promoter. In addition, sequence analysis has revealed fragments of human endogenous retroviruses inserted downstream from the C-alpha genes and at least two Alu repeat sequences. In collaboration with Drs. Zelazowski and Snapper of USUHS, we are examining the role of the Ku-DNAPK complex in isotype switching using a murine B cell line transfected with an expression construct designed to generate antisense RNA.
