Chromatin Architecture near a Potential 3′ End of the Igh Locus Involves Modular Regulation of Histone Modifications during B-Cell Development and In Vivo Occupancy at CTCF Sites

Abstract

The murine Igh locus has a 3′ regulatory region (3′ RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3′ RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3′ RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro- and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. Histone modifications were similar in resting splenic B cells and in splenic B cells induced by lipopolysaccharide to undergo CSR. From the pro-B-cell stage onward, the ∼11-kb region immediately downstream of hs4 displayed H3 and H4 modifications indicative of open chromatin. This region contained newly identified DNase I-hypersensitive sites and several CTCF target sites, some of which were occupied in vivo in a developmentally regulated manner. The open chromatin environment of the extended 3′ RR in mature B cells was flanked by regions associated with dimethylated K9 of histone H3. Together, these data suggest that 3′ RR elements are located within a specific chromatin subdomain that contains CTCF binding sites and developmentally regulated modules.

ACKNOWLEDGMENTS

This work was supported by NIH grant AI13509 (B.K.B.), NIH training grant T32 CA09173 (A.V.E.), an Albert Einstein College of Medicine Cancer Center Core grant P30CA13330, NIH grant AI30653 (L.A.E.), and NIH intramural funding (V.V.L.). F.E.G. was supported by NCI 5 F31 CA76942, a UNCF/MERCK Graduate Science Research Dissertation fellowship, and a National Medical Fellowship in Academic Medicine for Minority Medical Students. P.F. was a 2003 summer graduate student with V.V.L.

We thank Nasrin Ashouian, Alexa Price-Whelan, Huafeng Xie, and Nydiaris Hernandez for technical assistance and all members of the Lobanenkov and Birshtein laboratories for helpful discussions. We also thank Matthew Scharff, Ari Melnick, Ziqiang Li, Herbert Morse III, Svetlana Pack, and Dmitry Fyodorov for critical review of the manuscript. Our appreciation is extended to Kathryn Calame, Ranjan Sen, Arthur Skoultchi, Michael Krangel, and Anton Krumm for kind gifts of reagents and to Dipanjan Chowdhury, Brian Dynlacht, Katrina Morshead, Caroline Woo, Joseph Locker, Ari Melnick, Tomas Stopka, and Wendy Gombert for helpful advice.