178 | Chapter 7 in Jk accessibility. However, inactivation of SpiC, alone was not sufficient to provoke a breach of allelic exclusion77. Next, we explored the possible involvement of other transcription factors in the DNA damage-driven regulation of RAG1/2. The transcription factor B-cell leukemia 11a (BCL11a) is known to be a component of the transcriptional network regulating RAG expression. The major isoform of BCL11a, the BCL11a-XL, was shown to bind directly to the RAG1 promoter as well as to regulatory regions of other transcription factors involved in regulating RAG1 and RAG2, such as IKZF1, FOXO1, PAX5, Transcription Factor 3 (TCF3), SPIB, and interferon regulatory factor 4 (IRF4). Moreover, BCL11A was found to bind directly to the surrogate light chain VPREB1 promoter region.78 In our study, the NCS-induced DNA damage in human BCR-ABL1+ pre-B cells did not have any effect on the protein levels of BCL11a. However, whether the DNA breaks modulate the ability of BCL11a to bind to RAG1 promoter, has not been investigated. Recently, growth factor–independent 1b (Gfi1b) has been demonstrated to be a transcriptional repressor of RAG. Using mouse v-Abl cell lines, the repression of RAG transcription was achieved by direct binding of Gfi1b to the 5’ of the Erag, causing epigenetic changes in the Rag locus, as well as through repression of FOXO1 by Gfi1b79. Whether DNA damage triggers the binding of Gfi1b to the Erag enhancer has not been investigated, leaving a possibility of Gfi1b’s contribution to the RAG downregulation in response to genotoxic stress. Chapter 5 reveals that the NF-κB pathway plays an important role in the repression of RAG activity in pre-B cells. Several layers of regulations limit the expression of RAG1/2 in pre-B cells to the G1 phase of the cell cycle. The inappropriate activity of RAG1/2 during the S phase can contribute to chromosomal translocations, deletions, and other genomic alterations, ultimately leading to cellular dysfunction and disease development. Also, the RAG1/2 expression and activity in the S phase have previously been linked to an increase in genomic instability and lymphomagenesis80. In the course of B-cell development, there is a strict separation between pre-B cell proliferation and RAG expression/activity, serving as a safeguard against genomic instability81. Following the successful Igh recombination, the developing B cells at this stage are rather large, they divide fast and are in cycle. Before moving on to the next developmental stage, the cells go out of cycle, become smaller, re-activate expression of RAG1/2, and initiate recombination on the Igl82. In our study83, we used a mouse v-Abl-transformed pre-B cell line, where the Abelson virus introduces a developmental block and keeps the cells at large cycling pre-B cells stage. Notably, in the large cycling v-Abl transformed pre-B cells do not express RAG and their Igk or Igl is still in the germline configuration. Treatment with Abelson kinase inhibitor STI571 alleviates the differentiation block and induces a G1 arrest, at which the cells upregulate RAG1/2 and initiate Igl gene recombination9,81. In the cycling pre-B cells, AKT has been shown to repress the FOXO1 expression84. Our study showed
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