We studied the molecular mechanism of demethylation and its role in κ chain gene regulation. Following transfection into B cell cultures, this gene undergoes regional demethylation in a process that is developmentally regulated in a lineage- and stage-specific manner. Although a germline Vκ promoter is not required for the demodification activity, a fragment containing the intronic κ chain transcriptional enhancer and the nearby matrix attachment region is essential. In its natural location downstream to the Jκ5 sequence, this element induces bidirectional demodification of plasmid constructs in a distance- and orientation-independent manner. When this enhancer is placed in an upstream position, however, the κ gene remains modified and transcriptionally inactive, demonstrating that demethylation is required for κ chain activation. These studies suggest that the κ enhancer plays a dual role in regulating B cell differentiation by inducing demethylation and by promoting tissue-specific transcription.
Bibliographical noteFunding Information:
We are grateful to K. Rajewsky for making his data available to us prior to publication and to the members of the F. Alt laboratory for their helpful comments. We also acknowledge the assistance of G. Hirst in preparing the manuscript. This research was supported by a career development award from the Israel Research Fund (Y. B.) and by grants from the Israel Science Foundation (Y. B.), the Ehrfich Center for the Study of White Cells (Y. B.), the National Institutes of Health (H. C.), and the Israel Cancer Research Fund (H. C).