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(δ)1 and V(δ)2), diversity (D(δ)3), joining (J(δ)1 and J(δ)3), and constant (C(δ)) gene segments. Previous data indicated that with the TCR δ enhancer (E(δ)) present in the J(δ)3-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(δ)3 and 5' of J(α)1. 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(δ)1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of D(δ)3 was unaffected. We interpret these results to indicate that E(δ) is required for J(δ)1 but not D(δ)3 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(δ)3 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(δ)3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.