60 | Chapter 3 Results Experimental model and accumulation of RAG-dependent NBS1 peaks at Igk locus The mouse v-Abl/Bcl2-transformed Artemis -/- (AH2) and Artemis -/-Rag2-/- (R2K.Art) pre-B cells were used as a model to study the (off-)targeting capacity of RAG1/2 endonuclease. Transformation of B cells with Abelson murine leukemia virus arrests B-cell development in the early pre-B cell-like stage at which the expression of Rag1 and Rag2 is low and the light chain recombination minimal. Treatment with Abelson kinase inhibitor STI571 alleviates the developmental block, resulting in an increase in Rag1 and Rag2 mRNA expression. The subsequent increase in RAG1/2 endonuclease activity leads to Igk recombination. In this experimental model, Vk6-23/Jk1 recombination was monitored by PCR (Figure 1A). In time, the Vk6-23/Jk1 PCR signal increases (Figure 1B), suggesting more cells have undergone successful recombination Igk recombination. RAG cleavage creates pairs of coding and signal DNA ends, which are processed and joined by the canonical non-homologous end-joining (c-NHEJ) pathway of DNA DSB repair30. The DSB formation results in recruitment of the MRN complex (composed of MRE11, RAD50, and NBS1)31. The Artemis deficiency in v-Abl pre-B cells treated with STI571 enabled detection of of unrepaired DSB as Artemis functions as an endonuclease providing a hairpin opening activity32. Using this model, we took advantage of DSB accumulation, and thus accumulation of MRN members including NBS1 near the DSBs. Importantly, based on the experiments including wildtype v-Abl-transformed mouse pre-B cells, Artemis deficiency proved to be instrumental in the robust detection of RAG1/2-dependet DSBs (data not shown). The suitability of the model to interrogate RAG targeting was tested by assessing the binding of NBS1 protein to Jk regions by chromatin immunoprecipitation (ChIP) of NBS1 at Jk1, Jk2 and Jk5, and normalized to binding to Plac8 intronic region, used as a control region, Figure 1. (A) Wildtype (WT), Rag2-/- (R2K3), Artemis-/- (AH2) and Artemis-/-Rag2-/- (R2K.Art) v-Abl cells were treated with 5µM STI571 for 72h. Following the treatment, the recombination products of RAG1/2 activity were confirmed by assessing the presence of Vk6-23/ Jk1 recombination with the means of PCR. The PCR products were analyzed on agarose gel stained with ethidium bromide. GAPDH was used as a housekeeping gene. (B) Wildtype v-Abl cells were treated with 5µM STI571 for 6h, 12h, 24h, 48h, and 72h. The presence of Vk6-23/Jk1 recombination can be detected as early as 24h following the STI571 treatment with the means of PCR. The PCR products were analyzed on agarose gel stained with ethidium bromide. GAPDH was used as a housekeeping gene. (C) Binding of NBS1 protein to DNA was assessed by chromatin-immunoprecipitation (ChIP). Even though no productive recombination takes place in Artemis-/- cells (AH2), RAG1/2 endonuclease still creates DSBs at the Igκ locus, thus prompting the NBS1 binding to the vicinity of the DSBs. PCR was performed on the precipitated material using Jk1, Jk2, and Jk5 primer sets. The signal from NBS1 ChIP was normalized to the signal from Histone 4 (H4) ChIP, where H4 was used as a control antibody, its relative presence in both cell lines should be similar. PCR at Plac8 was used as a negative control, this is not active in developing B cells and no accumulation of DNA breaks is expected here.
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