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Locus1,2
* Division of Monoclonal Antibodies and
Division of Therapeutic Proteins, Food and Drug Administration, Center for Drug Evaluation and Research, Rockville, MD 20852; and
Department of Immunology and Molecular Pathology, Division of Infection and Immunity, University College London, London, United Kingdom
The 3-megabase Ig
locus undergoes differentially controlled nuclear positioning events and chromatin structural changes during the course of B cell development. The temporal association of chromatin structural changes, transcription, and recombination at the Ig locus was determined in a murine pre-B cell line that can be induced to recombine at the Ig locus and in ex vivo-cultured murine pre-B cells. Additionally, the timing of nuclear positioning relative to the temporal order of chromatin structural changes and recombination and transcription was determined. We demonstrate that before induction, the Ig locus was poised for recombination; both alleles were in a contracted state, and the enrichment of histone modifications and germline transcripts of specific V genes were observed. Histone modifications of the V genes did not vary upon induction but the levels of modifications correlated with the levels of germline V gene transcripts and recombination. Upon induction, but before VJ recombination, centromeric recruitment of single Ig alleles occurred. DNase I sensitivity of the entire locus increased gradually over the course of differentiation while the enrichment of histone modifications downstream of the V genes was increased in the silencer regions upstream of J1, within the Ig sterile transcript, the constant region, the Ei and E3' enhancers, and the recombining sequence. The ex vivo pre-B cells showed similar patterns of histone modifications across the locus except at the V genes. In this study, H3 acetylation correlated with levels of germline transcripts while H3 methylation correlated with levels of recombination.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Intramural Research Program of the Center for Drug Research and Review, Food and Drug Administration. J.S. was supported by a Wellcome Trust Project Grant.
2 Opinions expressed in this publication reflect the professional views of the authors and should not be viewed as official policy of the U.S. Food and Drug Administration or the government of the United States.
3 Current address: Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016.
4 Address correspondence and reprint requests to Dr. Marjorie Shapiro, Division of Monoclonal Antibodies, U.S. Food and Drug Administration, Center for Drug Evaluation and Research, 5600 Fishers Lane, Rockville, MD 20857. E-mail address: marjorie.shapiro{at}fda.hhs.gov
5 Abbreviations used in this paper: HC, H chain; LC, L chain; ts, temperature sensitive; ChIP, chromatin immunoprecipitation; Mb, megabase; Sis, silencer intervening sequence; RS, recombining sequence; FISH, fluorescence in situ hybridization; RSS, recombination signal sequence; RIC, RS information content score; Ct, threshold cycle; BAC, bacterial artificial chromosome.
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