182 | Chapter 7 when overexpressing FOXP1 in mouse v-Abl pre-B cells. We speculate that the BCL6-driven inhibition of p53 might have consequently limited the expression of miR-34a and alleviated the miR-34a-driven repression of the RAG1/2 transcription factor FOXP1. Collectively, our data, and the data of others, suggest that activation or stabilization of p53 suppresses gene recombination in developing B cells, thereby maintaining genomic stability. FOXP1 is known RAG1/2 transcription factor and has been shown to bind to Erag enhancer to regulate the expression of Rag1 and Rag2113. In our study, expression of Foxp1 mRNA and protein was rapidly downregulated after induction of the DNA damage in the form of g-irradiation, in v-Abl mouse pre-B cells treated with STI571; the absence of p53 partially prevented the downregulation of Foxp1 following the induction of DNA damage. Interestingly, a recent study by Cerna et al showed that also in chronic lymphocytic leukemia (CLL) cells, activation of the DDR resulted in rapid downmodulation of FOXP1. Furthermore, the study of Cerna et al provided compelling evidence that the p53/miR-34a pathway is involved in the regulation of FOXP1 levels in response to DNA damage. Silencing FOXP1, or overexpressing miR-34a mimics was shown to result in lower protein levels of phospho-AKT and phospho-ERK after BCR-crosslinking, thereby contributing to BCR signaling modulation in CLL cells105. Downregulation of miR-34a has been observed in various types of cancers and its expression level profiling holds promise as a prognostic indicator, as low miR-34a levels were associated with poor patiets’outcome114. Though some studies showed a clear decrease in miR-34a transcripts in cell lines lacking p53 or containing a mutant p53115,116, in other studies the correlation between p53 status and miR-34a expression was not established117. In the HCT116 colon cancer cell line miR-34a has been identified as a direct transcriptional target of p53 in response to genotoxic stress. Expression profiling further revealed that transcripts of genes involved in cell proliferation were significantly downregulated, whereas transcripts of genes involved in DNA repair and DNA checkpoints were significantly upregulated following the retroviral overexpression of miR-34a117. In the context of B cells, in human (NALM-6) and murine (70Z/3) pre-B cells lines and mouse bone marrow cells, the viral overexpression of miR-34a resulted in repression of Foxp1 and displayed an increase in CD19+c-kit+IgM- pre-B cells, while the levels of B220+CD43-IgM- pre-B cells were significantly reduced, suggesting that the miR-34a overexpression caused a partial block of B-cell development at the pre-to-pre-B cell stage. In fact, this study identified FOXP1 to be a bona fide target of miR-34a, and overexpression of the Foxp1 gene lacking the 3’-UTR rescued the miR-34a-induced B-cell developmental block118. This conflicting evidence might be related to the intrinsic variability between the used cell line models and cell types. Our data suggest that a regulatory circuit, involving p53/miR-34a/FOXP1 might exist in pre-B cells, where the DNA breaks drive expression levels of miR-34a in mouse primary pre-B cells that are undergoing light-chain recombination. Moreover, a decrease of miR-34a levels using the miR-34a sponge resulted in an increase of surface IgM+ in B-cells after the
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