MUTATIONS OF THE INTRONIC IGH ENHANCER AND ITS FLANKING SEQUENCES DIFFERENTIALLY AFFECT ACCESSIBILITY OF THE J(H)-LOCUS

To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (Emu) in generating accessibility of the J(H) locus for VDJ recombination, we generated ES cells in which Eu or its flanking sequences were mutated by replacement with or insertion of an expressed neo(r) gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre-B cell tines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A-MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core Emu [which includes part of the 3' matrix associated region (MAR) and the Imu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neo(r) gene into the J(H)-Cmu intron does not affect J(H) accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the Emu resulted in a dramatic cis-acting inhibition of rearrangement, demethylation and germline transcription of the associated J(H) locus. Surprisingly, insertion of the neo(r) gene into the 5' MAR sequence approximately 100 bp upstream of the core Emu also dramatically decreased recombination of the linked J(H) locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the Emu and upstream flanking sequences is required for efficient rearrangement of the J(H) locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.