Effect of CpG methylation on RAG1/RAG2 reactivity: implications of direct and indirect mechanisms for controlling V(D)J cleavage

It has been suggested that DNA methylation/demethylation is involved in regulating V(D)J rearrangement. Although methylated DNA is thought to induce an inaccessible chromatin structure, it is unclear whether DNA methylation can directly control V( D) J recombination independently of chromatin structure. In this study, we tested whether DNA methylation directly affects the reactivity of the RAG1/RAG2 complex. Specific methylation within the heptamer of the recombination signal sequences (RSS) markedly reduced V( D) J cleavage without inhibiting RAG1/RAG2 -DNA complex formation. By contrast, methylation at other positions around the RSS did not affect the reactivity of the RAG proteins. The presence of a methyl-CpG binding-domain protein inhibited the binding of the RAG1/RAG2 complex to all the methylated CpG sites that were tested. Our findings suggest that DNA methylation around the RSS may have a previously unexpected function in regulating V( D) J recombination by directly inhibiting V( D) J cleavage, in addition to its general function of inducing an inaccessible chromatin configuration.