152 | Chapter 6 The transcription factor FOXP1 was shown to regulate RAG1 and RAG2 expression by binding to the Erag enhancer. The bone marrow from Foxp1-/- mice showed impaired B-cell development, as the percentages of pro–B cells (IgM-B220+CD43+) and pre–B cells (IgM–B220+CD43–) were significantly lower than in Foxp1+/- bone marrows12. In our study, the mRNA expression of Foxp1 was decreased by 45.5% in g-irradiated WT primary pre-B-cell cultures undergoing light chain recombination following IL7 withdrawal, while the Foxp1 expression in Tp53-/- primary pre-B cells remained unaffected by irradiation (Figure 1G). In the mouse A70 pre-B cell line, v-Abl creates a developmental block, arresting the B cells in a stage at which the RAG1/2 expression is low and the Igk is not yet recombined. Treatment with STI571, an Abelson-kinase inhibitor, alleviates this block, induces RAG1/2 expression and Igk recombination13. In agreement, in STI571-treated mouse v-Abl-transformed pre-B cells, 5Gy g-irradiation or treatment with the p53-stabilizing agent Nutlin-3 resulted in the downregulation of RAG1 and FOXP1 protein expression, which was prevented by pretreatment with the ATM kinase inhibitor KU55933 (Figure 1H). The downregulation of RAG1 and FOXP1 following g-irradiation was not caused by activation of caspases, as shown by pretreatment with the pan-caspase inhibitor Z-VAD-FMK, nor by cell death, as shown by the amount of sub-G1 phase cells analyzed by FACS for DNA-content (propidium-iodide). (Suppl Figure 2A-B). Figure 2. (A) Western blotting analysis of FOXP1, BCL6, and b-actin (loading control) in mouse v-Abl WT (A70) cells transduced with either empty vector (LZRS), human full-length FOXP1 (FOXP1 FL), the isoform of human of FOXP1 (FOXP1 iso), or human BCL6. (B) The mouse v-Abl WT cells (A70) were transduced with either empty vector (LZRS), human full-length FOXP1 (FOXP1 FL), the isoform of human of FOXP1 (FOXP1 iso), or human BCL6. Control, untransduced A70 cells treated with STI571 for 72h to induce RAG1/2 expression and Igk recombination. The formation of coding joints following the treatment with STI571 was assessed by qualitative PCR analysis of one particular joint: Vκ6-23 to Jκ124 (bands representing coding joints in transduced cells are indicated by arrows). The PCR products were run in 2% agarose gel containing ethidium bromide. (C) Representative FACS staining of WT primary bone marrow cells, transduced either with pCL-ECO-miR-34a-sponge or with pCL-ECO-scrambled as a control. The cells were cultured for 7 days on OP9, Flt3L, and IL7, the amount of B220+IgM+ pre-B cells was measured 48h following the withdrawal of Flt3L and IL7 from the medium. (D) Quantification of the amount of B220+IgM+ pre-B cells by FACS staining as described in 2B (n=2, mean±SD, **p<0.05). (E) RT-PCR analysis of the relative miR-34a expression in primary WT bone marrow pre-B cells, initially cultured 7 days on OP9, Flt3L and IL7, then the Flt3L and IL7 were withdrawn from the medium for 72h and the cells were exposed to either 5 Gy or 0.5 Gy of g-irradiation followed by 2h of recovery, or treated with p53 stabilizer Nutlin-3 (2.5mM) for 4 hours. Control cells were treated with DMSO (n=2, mean±SD, **p<0.05). (F) Schematic representation of the proposed self-regulating negative feedback loop. In response to excessive DNA damage ATM is phosphorylated which in turn activates the p53 pathway. Subsequently, one of the consequences of p53 activation of miR-34a, which is a negative regulator of FOXP1, the RAG1/2 transcription factor. This might represent a mechanism by which the B cells protect themselves against excessive DNA damage, and dysregulation of this pathway might contribute to the genetic instability in developing B cells.
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