Long-Range Interactions between Three Transcriptional Enhancers, Active Vκ Gene Promoters, and a 3′ Boundary Sequence Spanning 46 Kilobases

Abstract

The mouse immunoglobulin kappa (Igκ) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igκ alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vκ gene promoters. In addition, the 5′ region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3′ with 3′ boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the β-globin locus and suggest a dynamic modulation of the spatial organization of the active Igκ locus. Chromatin immunoprecipitation experiments reveal that the interacting Igκ gene cis-acting sequences are associated with AP-4, E47, and p65NF-κB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-κB still exhibit mutual interactions between the Igκ gene enhancers.

ACKNOWLEDGMENTS

This investigation was supported by grants GM29935 and GM59809 from the National Institutes of Health and grant I-823 from the Robert A. Welch Foundation.

We thank Piotr Widlak for a critical review of the manuscript and Alisha Tizenor for graphic illustrations.