Mechanisms that direct ordered assembly of T cell receptor β locus V, D, and J gene segments

Mechanisms that direct ordered assembly of T cell receptor β locus V, D, and J gene segments

^*Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School and Center for Blood Research, Boston, MA 02115; and ^†Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110

Contributed by Frederick W. Alt

Abstract

T cell receptor (TCR) β variable region genes are assembled in progenitor T cells from germ-line Vβ, Dβ, and Jβ segments via an ordered two-step process in which Dβ to Jβ rearrangements occur on both alleles before appendage of a Vβ to a preexisting DJβ complex. Direct joining of Vβ segments to nonrearranged Dβ or Jβ segments, while compatible with known restrictions on the V(D)J recombination mechanism, are infrequent within the endogenous TCRβ locus. We have analyzed mechanisms that mediate ordered Vβ, Dβ, and Jβ assembly via an approach in which TCRβ minilocus recombination substrates were introduced into embryonic stem cells and then analyzed for rearrangement in normal thymocytes by recombinase-activating gene 2-deficient blastocyst complementation. These analyses demonstrated that Vβ segments are preferentially targeted for rearrangement to Dβ as opposed to Jβ segments. In addition, we further demonstrated that Vβ segments can be appended to nonrearranged endogenous Dβ segments in which we have eliminated the ability of Dβ segments to join to Jβ segments. Our findings are discussed in the context of the mechanisms that regulate the ordered assembly and utilization of V, D, and J segments.

Footnotes

↵ ‡ Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305.
↵ § Center for Cancer Research, Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139.
↵ ¶ To whom reprint requests should be addressed. E-mail: alt{at}rascal.med.harvard.edu.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.130190597.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.130190597
Abbreviations:

TCR,

T cell receptor;

RAG,

recombinase-activating gene;

V,

variable;

D,

diversity;

J,

joining;

RDBC,

RAG-2-deficient blastocyst complementation;

RSS,

recombination signal sequence, ES, embryonic stem