Role of the Ubiquitin Proteasome System in the Regulation of V(D)J Recombination

Abstract

V(D)J recombination, the only known site specific DNA rearrangement in vertebrates, is the process that is responsible for the development of vertebrate adaptive immunity. It mediates the assembly of antigen receptor genes from component variable (V), diversity (D) and joining (J) gene segments interspersed by non-coding DNA. Lymphoid specific RAG1 and RAG2 proteins together form the “recombinase” that initiates the recombination event to rearrange the dispersed gene segments into a contiguous V(D)J region encoding the variable portion of an antigen receptor. In addition to its DNA binding and cleavage activities, RAG1 also harbors a ubiquitin ligase (E3) activity. Mutations in RAG1 that abrogate its ubiquitin ligase activity have been identified in patients with various immune deficiencies. Thus, RAG1-dependent ubiquitylation(s) may be important in regulating V(D)J recombination. Other RAG1 independent ubiquitylation events are also known to influence the regulation of V(D)J recombination.

This work demonstrates that 26S proteasome activity is required to promote an early step in V(D)J recombination, while ubiquitylation(s) supported by the ubiquitin activating enzyme (E1) is required throughout recombination, as assessed in cultured cells treated with 26S proteasome and E1 inhibitors. Furthermore, the inhibitor treatments revealed that both 26S proteasome and E1 activities were required prior to DNA cleavage. Treatment with stage-specific cell cycle inhibitors demonstrated that recombination did not require transit through cell cycle, indicating that the recombination defects upon proteasome or E1 inhibition were not due to indirect effects on cell cycle progression. These data suggest that a ubiquitin-dependent degradation event is a pre-requisite at an early stage, possibly for recombinase assembly or activation, while other ubiquitylation events are required later in recombination.

This work also identifies two ubiquitin conjugating enzymes (E2s) namely, UBE2A and CDC34, required for in vitro chromosomal recombination by using dominant negative forms of these and other E2 enzymes. Thus, certain E2 enzymes are specifically important in promoting robust recombination. Finally, ubiquitin ligase deficient mutants of RAG1 were found to be impaired in supporting DNA cleavage. Taken together, the findings in this study are consistent with the hypothesis that the degradation of a negative regulator is essential to enable efficient V(D)J recombination.