Enhancer control of V(D)J recombination at the TCRbeta locus: differential effects on DNA cleavage and joining.
Deletion of the TCRbeta transcriptional enhancer (Ebeta) results in nearly complete inhibition of V(D)J recombination at the TCRbeta locus and a block in alpha beta T cell development. This result, along with previous work from many laboratories, has led to the hypothesis that transcriptional enhancers affect V(D)J recombination by regulating the accessibility of the locus to the recombinase. Here we test this hypothesis by performing a detailed analysis of the recombination defect in Ebeta-deleted (Ebeta-/-) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flanking Dbeta and Jbeta gene segments in Ebeta-/- thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCRbeta locus. These sites are also subject to in vitro cleavage by the V(D)J recombinase in both Ebeta-/- and Ebeta+/+ thymocyte nuclei. However, the corresponding Dbeta-to-Jbeta coding joints are further reduced (by 100- to 300-fold) in Ebeta-/- thymuses. Formation of extrachromosomal Dbeta-to-Jbeta signal joints appears to be intermediately affected and nonstandard Dbeta-to-Dbeta joining occurs at the Ebeta-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCRbeta recombination in the absence of the Ebeta element and suggest an additional function for Ebeta in the process of DNA repair at specific TCRbeta sites during the late phase of the recombination reaction.
Centre d'Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille-Luminy, Marseille Cedex 9, France.
W M
WM
Hempel
P
P
Stanhope-Baker
N
N
Mathieu
F
F
Huang
M S
MS
Schlissel
P
P
Ferrier
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D011995:Recombination, Genetic; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D045567:VDJ Recombinases
D017931:DNA Primers; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases