102 | Chapter 4 Taken together, these results suggest that DNA breaks that originate from RAG1/2 activity and Ig gene rearrangements are involved in the down-regulation of Rag1 mRNA expression in ATM-dependent manner. Moreover, it suggests that the kinase function of ATM restricts the activity of the RAG1/2 endonuclease complex. To directly assess the impact of ATM kinase on RAG1/2 activity we made use of a novel fluorescent reporter system that has an antisense green fluorescent protein (GFP) cassette, which is flanked by a 12-basepair spacer RSS and a 23-basepair spacer RSS26. Functional expression of the RAG1/2 complex mediates inversional recombination that results in the sense orientation of the GFP cassette, which can be monitored in cells that co-express control red fluorescent protein (RFP) (Figures 7A, 7B). Treatment of mouse v-Abl cells with KU55933 resulted in a modest but reproducible increase in spontaneous (Figure 7C upper panels and Figure 7D) and STI571-induced RAG-dependent reporter activity (Figure7C lower panels and Figure 7E), consistent with a modest increase in Igh allelic inclusion in ATM-deficient mouse splenic and bone marrow B cells46. Collectively, these results show that ATM kinase activity is involved in limiting the activity of the RAG1/2 complex. Discussion The strict and ordered regulation of the RAG1/2 complex in developing B cells ensures the expression of a functional B-cell receptor required for B-cell maturation, while minimizing the risk of collateral damage emanating from this endonuclease. As there is ample evidence for the involvement of illegitimate RAG1/2 activity in genomic alterations found in mouse and human B-cell malignancies15-17,47, a thorough understanding of the mechanisms that control RAG1/2 expression will provide insight into the etiology of these malignancies and may even yield strategies to curb this potentially hazardous endonuclease. Previously, it was shown that RAG1/2 activity is restricted in a cell-cycle dependent manner48, which involves the cyclinA/CDK2-triggered proteasomal destruction of the RAG2 protein15, whereas RAG1 protein stability does not appear to be cell-cycle regulated13. At the transcriptional level, the expression of the RAG1 and RAG2 genes, residing in the same locus, is coordinately regulated by the binding of several transcription factors to cis-acting sequences12, of which FOXO1 and FOXP1 binding to the Erag enhancer emerge as major players in the regulation of RAG expression in B lymphocytes32,49. In this study, we investigated whether additional mechanisms, other than cell cycle-dependent RAG2 protein degradation, are involved limiting RAG-dependent DNA damage. In agreement with previous studies, we find that the DNA damage response regulates RAG in normal and malignant pre-B cells46,50. We show that DNA damage induces a rapid ATM-dependent down-regulation of RAG1 and RAG2 mRNA and protein expression. Our finding that recombinant expression of RAG1 in HEK293 cells was not affected by DNA damage suggests a predominantly transcriptional regulatory mechanism, although in
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