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Productive Coupling of Accessible Vβ14 Segments and DJβ Complexes Determines the Frequency of Vβ14 Rearrangement¹

Sheila Ranganath^*, Andrea C. Carpenter, Megan Gleason^*, Albert C. Shaw^2,*, Craig H. Bassing^3, and Frederick W. Alt^3,*

^* Howard Hughes Medical Institute, The Children’s Hospital, CBR Institute for Biomedical Research, and Department of Genetics, Harvard University Medical School, Boston, MA 02115; and Immunology Graduate Group, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Abramson Family Cancer Research Institute, Philadelphia, PA 19104

To elucidate mechanisms that regulate Vβ rearrangement, we generated and analyzed mice with a V(D)J recombination reporter cassette of germline Dβ-Jβ segments inserted into the endogenous Vβ14 locus (Vβ14^Rep). As a control, we first generated and analyzed mice with the same Dβ-Jβ cassette targeted into the generally expressed c-myc locus (c-myc^Rep). Substantial c-myc^Rep recombination occurred in both T and B cells and initiated concurrently with endogenous Dβ to Jβ rearrangements in thymocytes. In contrast, Vβ14^Rep recombination was restricted to T cells and initiated after endogenous Dβ to Jβ rearrangements, but concurrently with endogenous Vβ14 rearrangements. Thus, the local chromatin environment imparts lineage and developmental stage-specific accessibility upon the inserted reporter. Although Vβ14 rearrangements occur on only 5% of endogenous TCRβ alleles, the Vβ14^Rep cassette underwent rearrangement on 80–90% of alleles, supporting the suggestion that productive coupling of accessible Vβ14 segments and DJβ complexes influence the frequency of Vβ14 rearrangements. Strikingly, Vβ14^Rep recombination also occurs on TCRβ alleles lacking endogenous Vβ to DJβ rearrangements, indicating that Vβ14 accessibility per se is not subject to allelic exclusion.

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 National Institutes of Health Grant AI20047 (to F.W.A.) and the Department of Pathology of the Children’s Hospital of Philadelphia (to C.H.B.). S.R. was supported by a Genentech/IDEC Fellowship from the American Cancer Society and the Dana Farber Cancer Institute Postdoctoral Training Program in Cancer Immunology. A.C.C. was supported by the Training Program in Immune System Development and Regulation at the University of Pennsylvania. C.H.B. is a Pew Scholar in the Biomedical Sciences. F.W.A. is an Investigator of the Howard Hughes Medical Institute.

² Current address: Section of Infectious Diseases, Yale University School of Medicine, New Haven, CT 06520.

³ Address correspondence and reprint requests to Dr. Craig H. Bassing, Children’s Hospital of Philadelphia, Abramson Research Center, Room 807A, 3615 Civic Center Boulevard, Philadelphia, PA 19104. E-mail address: bassing{at}email.chop.edu or Dr. Frederick W. Alt, The Children’s Hospital, Karp Research Building, Room 9216, 1 Blackfan Circle, Boston, MA 02115. E-mail address: alt{at}enders.tch.harvard.edu

⁴ Abbreviations used in this paper: RSs, recombination signal sequence; DN, double negative; ES, embryonic stem; WT, wild type; RDBC, RAG2-deficient blastocyst complementation.