94 | Chapter 4 FOXO1 is involved in the DNA damage-induced regulation of RAG1 Based on these results we decided to study the effects of DNA damage on the transcriptional regulation of RAG expression in pre-B cells in further detail. FOXO1 is a crucial non-redundant transcription factor in developing B cells, and a key regulator of RAG1/2 expression at the pre-B cell stage32,33. Transcription of the RAG locus in B cells is controlled by the evolutionary conserved enhancer element Erag11. FOXO1 was shown to bind Erag in RAG-expressing small pre-B cells and is involved in driving RAG expression from this cis-acting sequence33,34. To further establish the involvement of FOXO1 in the DNA damage-induced regulation of RAG1 expression we assessed the binding of FOXO1 to the Erag enhancer following DNA damage, using chromatin immunoprecipitation (ChIP) (Figure 2C). We found that the binding of FOXO1 to the Erag enhancer was abolished by NCS treatment in BV173 and SUP-B15 cells (Figure 2D). Pre-treatment with the ATM kinase inhibitor fully prevented the loss of FOXO1-Erag binding upon induction of DNA damage. In addition, we found that phosphorylated ATM (Ser1981) was present at the Erag enhancer region in NCS-treated cells (Figure 2E), indicating that regulation of FOXO1-binding by ATM possibly could take place in the context of chromatin. Figure 3. FOXO1 protein is cleaved/degraded upon DNA damage. (A) Western blot analysis of FOXO1, AKT and mTOR phosphorylation in BV173 cells treated as indicated, the b-actin blot was used to control for loading. (B) Realtime quantitative RT-PCR analysis of FOXO1 mRNA expression in BV173 cells treated as indicated, means ± SEM are shown (upper graph, n=4, *** p=0.0003, one-sample t test), and Foxo1 mRNA in WT mouse IL-7 dependent pre-B cell cultures, cells were treated as indicated 48h after IL7 withdrawal, cultures from 4 mice were analyzed independently, means ± SEM are shown (lower graph, n=4, * p=0.02, one-sample t test).
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