Requirement for ubiquitin conjugation and 26S proteasome activity at an early stage in V(D)J recombination

Abstract

V(D)J recombination, the process that rearranges gene segments to assemble mature antigen receptor genes, relies on a recombinase comprising the RAG1 and RAG2 proteins. RAG1 is a multi-functional enzyme including DNA binding and cleavage as well as ubiquitin ligase activities, all of which appear to contribute to its role in recombination. Here we demonstrate that components of the ubiquitin conjugation machinery and the 26S proteasome are required for an early step in V(D)J recombination. Inhibitors of the 26S proteasome and ubiquitin activating enzyme (E1) blocked both chromosomal and extra-chromosomal recombination when added 1h following transfection/induction, but they had no effect when added 16 h later. There was no effect on expression of RAG1, and recombination did not require transit through the cell cycle, confirming that inhibition was not due to an indirect effect on cell cycle arrest or protein expression. Experiments in which RAG1 translation was blocked with cyclohexamide after 16 h of expression indicated that many active recombination complexes were formed within this window, although recombination products continued to accumulate for 48 h. These data suggest that ubiquitin-dependent degradation is an early step in complex assembly or activation, and are consistent with our previous hypothesis that degradation of a negative regulator is required to trigger recombination.

Fig. 1

Requirement for 26S proteasome for extra-chromosomal recombination. (A) Cells were treated with lactacycstin as described in Materials and Methods, and 26S proteasome activity was determined at the times indicated. (B) Cell viability was assessed by tryphan blue exclusion after 48 hours lactacystin treatment. (C) General scheme for all recombination experiments. Cells were transfected or induced to undergo recombination at time 0. For treated samples (filled bars), treatment was added at the time indicated and DNA was harvested 48 hrs. after transfection/induction. For control samples (white bars), DNA was harvested at the time indicated. Recombination assays were carried out on pJH200 (D) or pJH299 (E) as described in Materials and Methods in the absence of inhibitor (white bars) or in the presence of lactacycstin (black bars) or nocodazole (hatched bars). (B–E) depict average and standard deviation of at least three independent trials. (F) RAG1 and β-actin expression were analyzed by Western blot. Results are representative of multiple trials. (G) cells were treated with nocodazole as indicated and DNA content was determined by FACS analysis. (1) t = −9.99 [2], p = 0.01. (2) t = −16.6 [2], p = 0.004. (3) t = −5.08 [2], p = 0.037.

Fig. 2

Requirement for 26S proteasome for chromosomal recombination. (A) Recombination on the kappa locus in 103Bcl2-4 cells was analyzed as described in Materials and methods. White bars, cells were harvested after incubation at 39°C for the time indicated. Black bars, cells were incubated at 39°C for the time indicated then treated with 26S proteasome inhibitor then incubated at 39°C for a total of 48 hours. Average and standard deviation of three independent trials are shown. (B) RAG1 and β-actin expression were analyzed by Western blot in induced (39°C) and un-induced (34°C) samples. Duplicate trials are shown. PI, 26S proteasome inhibitor; EtOH, ethanol carrier control. (1) t = −4.32 [2], p = 0.05. (2) t = −10.6 [2], p = 0.009. (3) t = 1.67 [2], p = 0.237.

Fig. 3

Affect of protein synthesis inhibition on recombination. Recombination of pJH200 (A) or pJH299 (B) was assessed as described in Materials and Methods. White bars, untreated cells were harvested at the time indicated post-transfection. Grey bars, cells were treated with cyclohexamide at the time indicated post-transfection, and harvested 48 hours post-transfection. Average and standard deviation of three independent trials are shown. (C) RAG1 (R1) and β-actin expression were analyzed by Western blot. Results of triplicate trials are shown. (1) t = −4.99 [2], p = 0.038.

Fig. 4

Requirement for Ubiquitin Activating Enzyme (E1) for recombination. Recombination assays were carried out on pJH200 (A) or pJH299 (B) or on the kappa locus (C) as described in Materials and Methods in the absence of inhibitor (white bars) or in the presence of E1 inhibitor PYR-41 (grey bars). Average and standard deviation of three independent trials are shown. (D) RAG1 (R1) and β-actin expression in the presence of PYR-41 or DMSO carrier control were analyzed by Western blot. Results of duplicate trials are shown. (E) Recombination of pJH200 in E36ts20 CHO was assessed at permissive (30°C) and non-permissive (42°C) temperature. SJ-specific PCR product is indicated. (F) Semi-quantitative PCR of recombination products shown in (E).

Fig. 5

DNA replication is required for recombination on extra-chromosomal substrates. Recombination assays were carried out on pJH200 (A) or pJH299 (B) as described in Materials and methods in the absence of inhibitor (white bars) or in the presence of aphidicolin (hatched bars). (C) Recombination in 103-Bcl 2/4 cells is shown. Inhibitor was added as indicated 1 hour after shift to 39°C. For CJ (light bars) on the kappa locus, recombination was measured 48 hrs after shift to 39°C. For SJ (dark bars) recombination was measured after 48 hrs at 39°C followed by 24 hrs at 34°C. Recombination is shown as a percent of carrier control, ethanol for aphidicolin and DMSO for nocodazole.