ENHANCER CONTROL OF LOCAL ACCESSIBILITY TO V(D)J RECOMBINASE

We have studied the role of transcriptional enhancers in providing rec ombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delt a gene minilocus. This transgene is composed of unrearranged variable (V(delta)1 and V(delta)2), diversity (D(delta)3), joining (J(delta)1 a nd J(delta)3), and constant (C-delta) gene segments. Previous data ind icated that with the TCR delta enhancer (E-delta) present in the J(del ta)3-C-delta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rear rangement is intact, but VD-to-J rearrangement is inhibited. We propos ed that E-delta is necessary for J segment but not D segment accessibi lity and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype, In this study, we tested this notion by using ligation-mediated PCR to assess the fo rmation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of D(delta)3 and 5' of J(delta)1. In five li nes of mice carrying multicopy integrants of constructs that either la cked E-delta or carried an inactivated E-delta, the frequency of DSBs 5' of J(delta)1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of D(delta)3 was unaffected, We interpret these results to indicate that E-delta is required for J(de lta)1 but not D(delta)3 accessibility within the minilocus, and we con clude that enhancers regulate V(D)J recombination by providing local a ccessibility to the recombinase, cis-acting elements other than E-delt a must maintain D(delta)3 in an accessible state in the absence of E-d elta. The analysis of DSB formation in a single-copy minilocus integra nt indicates that efficient DSB formation at the accessible RSS 3' of D(delta)3 requires an accessible partner RSS, arguing that RSS synapsi s is required for DSB formation in chromosomal substrates in vivo.