4 DNA damage response regulates RAG1/2 expression through ATM-FOXO1 | 83 Introduction In developing lymphocytes, Ag receptors are assembled by recombination of immunoglobulin heavy chain (Igh) and light chain (Igl) variable (V), diversity (D), and joining (J) gene segments. This recombination process is initiated by the recombination activating gene (RAG) proteins RAG1 and RAG2, which form a heterodimeric complex possessing endonuclease capacity, creating double-stranded DNA breaks (DSBs) at recombination signal sequences (RSS) adjacent to the immunoglobulin (Ig) and T-cell receptor (Tcr) gene segments1,2. Subsequent to DNA cleavage, the RAG1/2 complex remains associated with the DNA ends to facilitate their repair by the non-homologous end-joining repair mechanism3. V(D)J recombination is initiated in a monoallelic fashion, and following a successful in-frame Igh V to DJ rearrangement in pro-B cells, RAG expression is rapidly down-regulated, thereby preventing biallelic recombinations and enforcing allelic exclusion. After productive Igh rearrangement the pre-B cell receptor (pre-BCR) is formed, expressed on the cell membrane, and as a result of pre-BCR expression, pre-B cells enter the cell cycle. At the pre-B cell stage, an interplay between IL-7 receptor (IL-7R) signaling and preBCR signaling coordinates subsequent B-cell development and RAG expression4. IL-7R activates phosphoinositide 3-kinase (PI3K), which via AKT, represses forkhead box protein O1 (FOXO1), a major non-redundant RAG1 and RAG2 transcription factor. Moreover, IL-7R activates STAT5 that, on one hand, drives cyclin D3 expression and promotes cell proliferation, and on the other hand limits accessibility of the Igk light chain locus5. The pre-BCR regulates pre-B cell proliferation by activating spleen tyrosine kinase (SYK) and RAS-extracellular signal-regulated kinase (ERK) signaling, which represses cyclin D3 and drives early region 2A (E2A), an important regulator of Igk locus transcription6,7. In addition, pre-BCR signals to pre-B cell linker (BLNK, also known as SLP-65), inhibiting the PI3K-AKT pathway and enhancing Igk accessibility by a redistribution of long-range chromatin interactions8,9. Attenuation of IL7 signaling in mouse pre-B cells drives B-cell differentiation and is associated with up-regulation of FOXO1, re-expression of RAG1 and RAG2, and Igk rearrangement4. RAG1 and RAG2 are convergently transcribed from the same locus. Cooperation of distant sequences in cis, the proximal enhancer, the distal enhancer, and the RAG enhancer (Erag), with RAG promoters is required for regulating RAG expression in lymphocytes10-12. Erag is the strongest enhancer and a complex network of transcription factors, including Foxo1, forkhead box protein P1 (FoxP1), paired box protein 5 (Pax5), nuclear factor kappalight-chain-enhancer of activated B cells (NF-kB), and Ikaros, was shown to activate RAG transcription by binding to this element12. Unlike RAG1, termination of RAG2 expression is linked to cell-cycle progression. In dividing cells, RAG2 expression and activity are detected in the G1-phase whereas it is absent in the S- and G2/M-phases of the cell cycle13. It was shown that phosphorylation
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