Activation of immunoglobulin mu gene expression involves stepwise demethylation.

In an attempt to identify stages of mu gene activation subsequent to VHDHJH assembly, we investigated two pre-B cell lines (A1 and A8) that have both alleles of the JH locus rearranged but do not make mu polypeptides. The block in A1 reflects incorrect VHDHJH assembly on both alleles. In A8, although an out-of-phase VHDHJH-C mu allele is transcribed, the properly assembled allele is silent, despite having a normal VH promoter and mu enhancer. Thus, transcription can be restricted to a single mu allele. Low level mu transcription in both lines was associated with demethylation of the VHDHJH and enhancer regions but not of the C mu gene. Markedly elevated mu transcription ensued on lipopolysaccharide stimulation or fusion to a plasmacytoma, but only fusion induced C mu demethylation. Hence stepwise demethylation is implicated in mu gene regulation, but enhanced expression can also occur independently of, or prior to, demethylation.