Enhancer Control of Local Accessibility to V(D)J Recombinase

Abstract

We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) _δ gene minilocus. This transgene is composed of unrearranged variable (V_δ1 and V_δ2), diversity (D_δ3), joining (J_δ1 and J_δ3), and constant (C_δ) gene segments. Previous data indicated that with the TCR _δ enhancer (E_δ) present in the J_δ3–C_δ 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 rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that E_δ is necessary for J segment but not D segment accessibility 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 formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3′ of D_δ3 and 5′ of J_δ1. In five lines of mice carrying multicopy integrants of constructs that either lacked E_δ or carried an inactivated E_δ, the frequency of DSBs 5′ of J_δ1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3′ of D_δ3 was unaffected. We interpret these results to indicate that E_δ is required for J_δ1 but not D_δ3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than E_δ must maintain D_δ3 in an accessible state in the absence of E_δ. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3′ of D_δ3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.