104 | Chapter 4 RAG locus and RAG expression, but not for the expression of several other FOXO1 target genes53. Unfortunately, we could not assess the effects of DNA damage on FOXO1 Ser215 phosphorylation, as antibodies recognizing this specific FOXO1 phospho-protein are not available. It was shown that MAPKAPK5 expression is required for RAG expression in pre-B cells, leaving open the possibility that MAPKAPK5 levels itself could be regulated by DNA damage. To address this possibility we assessed the levels of MAPKAPK5 in STI571-treated BV173 pre-B cells but found no difference in MAPKAPK5 levels upon DNA damage and/ or ATM kinase inhibition (SUPPL Figure 3). Additionally, it was shown that the BCL11a transcription factor activates RAG1/2 expression in pre-B cells and is critical component of the gene regulatory network controlling early B-cell development54. However, we neither found evidence for the DNA damage-mediated regulation of BCL11a in STI571-treated BV173 pre-B cells (SUPPL Figure 3). Based on the above we hypothesize that the ATM kinase may possibly function by recruiting or activating additional factors that regulate the DNA-binding capacity and stability of FOXO1 in RAG-expressing pre-B cells, of which the identity remains to be elucidated. The ATM kinase may be involved in post-translational modifications that affect the DNA-binding properties of FOXO1, other than phosphorylation. For instance, it was shown that acetylation of FOXO1 decreased its affinity for the target DNA55. The acetylation status of FOXO1 is regulated by the acetyltransferase C-AMP response element-binding protein (CBP) and the NAD-dependent histone deacetylase sirtuin 1 (SIRT1)56,57. Of interest, ATM was shown to activate the deleted in breast cancer 1 (DBC1) protein upon induction of DNA damage, which is a negative regulator of SIRT158,59. A possible scenario might be that ATM inhibits the deacetylation of FOXO1 by activating DBC1, thereby regulating the DNA-binding capacity of FOXO1. In an earlier study in A549 lung carcinoma cells, the acetylation of overexpressed FLAG-tagged FOXO1 could be detected upon induction of DNA damage58. However, we were unable to convincingly show acetylation of endogenous FOXO1 in BV173 cells exposed to NCS (data not shown). Consistent with earlier reports, we show that DNA breaks resulting from RAG1/2 activity trigger an ATM-dependent negative feedback loop that limits the expression and activity of RAG1/246,50. Previously, it was shown that this negative feedback regulation contributes to Igk allelic exclusion by suppressing secondary Vk to Jk rearrangements and therefore is an integral part of B-cell development46. In agreement with this, we find that the activity of RAG1/2 is slightly enhanced upon inhibition of the ATM kinase function. It was suggested that ATM regulates RAG1/2 expression by repressing the growth arrest and DNA-damage-inducible protein (GADD45a)46, which is important for RAG1/2 transcription in mouse v-Abl transformed pre-B cells32. However, we could not detect any alterations in the mRNA or protein levels of Gadd45a in mouse v-Abl transformed pre-B cells and BV173 BCR-ABL-positive B-ALL cells 2h after DNA damage induction by NCS, nor after (pre-) treatment of these cells with the ATM kinase inhibitor (SUPPL Figure 4).
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