92 | Chapter 4 Transcriptional regulation of RAG expression by DNA damage Exposure of cells to DNA-damaging agents may result in the activation of a caspase cascade and subsequent proteolysis31. Accordingly, we observed that the 2-hour NCS treatment activated caspase-3 (Figure 2A). To provide evidence that NCS-induced RAG1 protein down-regulation is not merely a result of proteolysis by activated caspases, BV173 cells were pre-treated with the pan-caspase inhibitors Q-VD-OPH or Z-VAD (data not shown) one hour prior to NCS treatment. Subsequent treatment with NCS resulted in RAG1 down-modulation even in the presence of Q-VD-OPH, whereas cleavage of pro-caspase-3 was efficiently inhibited (Figure 2A), showing that caspase-mediated proteolysis did not account for the loss of RAG1 protein following DNA damage. DNA damage significantly affected RAG1 mRNA and protein expression levels in pre-B cells, indicating that regulation takes place at the transcriptional level as well as the post-translational level. To further dissect the relative contribution of these regulatory mechanisms, we ectopically expressed RAG1 protein from a heterologous promoter (i.e., plasmid-driven) in HEK293 cells. Surprisingly, while treatments with increasing doses of ionizing radiation or increasing concentrations of NCS clearly elicited a DNA damage response, as shown by increased phospho-p53 (Ser15) levels, DNA damage did not affect RAG1 protein levels under these conditions, suggesting that DNA damage-mediated regulation of RAG1 protein requires its endogenous transcriptional regulation and that the post-translational regulation of RAG1 protein expression is not conserved between different cell-types (Figure 2B). Figure 2. RAG1 down-regulation upon DNA damage is caspase-3-independent, requires its endogenous transcriptional context, and coincides with loss of FOXO1-Erag binding. (A) Western blotting analysis of RAG1 and cleaved caspase-3 expression in STI571-treated BV173 BCR-ABL-positive B-ALL cells. Cells were pre-treated with 10 mM Q-VD-OPH 1h prior to 2h treatment with 50 ng/mL NCS treatment. The b-actin blot was used to control for loading. (B) Western blotting analysis of HEK293 cells transfected with pEBB-RAG1 encoding full-length mouse RAG1 (kind gift from Dr. M. van der Burg). Cells were treated as indicated and harvested 2h after induction of DNA damage. The phospho-p53 (Ser15) blot was used to show the induction of the DNA damage response. The b-actin blot was used to control for loading. (C) Schematic depiction of the human RAG1/2 locus, numbers indicate chromosomal coordinates and arrows indicate transcriptional direction. Erag enhancer and ChIP-PCR target region are shown. (D) Binding of FOXO1 and control rabbit IgG, and (E) phospho-ATM (Ser1981) and control mouse IgG to the Erag enhancer in STI571-treated BV173 cells and SUP-B15 BCR-ABL-positive B-ALL cells as determined by ChIP and expressed as percent of input DNA. Cells were treated as indicated. ChIP experiments were performed in triplicate. Statistical significances were determined by one-way analysis of variance (ANOVA) using a Bonferroni’s posttest, means ± SEM are shown (** p<0.01; *** p<0.001).
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