Dual mechanism of Rag gene repression by c-Myb during pre-B cell proliferation (original) (raw)
Related papers
Ebf1 and c-Myb Repress Rag Transcription Downstream of Stat5 during Early B Cell Development
The Journal of Immunology, 2013
The temporal control of RAG (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the IL-7R and precursor B cell Ag receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful Ig H chain gene rearrangement. Whereas the mechanism of RAG2 protein downregulation is well defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson murine leukemia virus-transformed B cells to model this stage of development, we identified early B cell factor 1 (Ebf1) as a strong repressor of Rag transcription. Short hairpin RNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb.
Nature Immunology, 2008
Regulated expression of the recombinase RAG-1 and RAG-2 proteins is necessary for generating the vast repertoire of antigen receptors essential for adaptive immunity. Here, a retroviral cDNA library screen showed that the stress-regulated protein GADD45a activated transcription of the genes encoding RAG-1 and RAG-2 in transformed pro-B cells by a pathway requiring the transcription factor Foxo1. Foxo1 directly activated transcription of the Rag1-Rag2 locus throughout early B cell development, and a decrease in Foxo1 protein diminished the induction of Rag1 and Rag2 transcription in a model of receptor editing. We also found that transcription of Rag1 and Rag2 was repressed at the pro-B cell and immature B cell stages by the kinase Akt through its 'antagonism' of Foxo1 function. Thus, Foxo1 is a key regulator of Rag1 and Rag2 transcription in primary B cells. Adaptive immunity depends on the concerted action of the lymphocyte-restricted products of recombination-activating gene 1 (RAG-1; A002009) and recombination-activating gene 2 (RAG-2; A002010), which catalyze the somatic DNA rearrangement of variable, diversity and joining gene segments forming the variable-domain exons of B cell antigen receptors (BCRs) and T cell antigen receptors (TCRs) 1. In B cells, RAG activity occurs in two discrete waves: first at the common lymphoid progenitor and pro-B cell stages during immunoglobulin heavychain locus rearrangement and then again at the pre-B cell stage during immunoglobulin lightchain locus rearrangement 2,3. Productive rearrangement of both heavy-and light-chain genes leads to BCR expression at the immature B cell stage. Basal signaling from a self-tolerant BCR limits RAG activity at this stage and ultimately leads to complete loss of expression of the genes encoding RAG-1 and RAG-2 (Rag1 and Rag2, respectively; collectively called 'Rag' here) as B cells mature further 4. If, however, the BCR recognizes self antigen, development halts and Rag expression continues, resulting in further light-chain locus rearrangement (receptor editing) and altered BCR specificity until an 'innocuous' BCR is expressed or the potential for light-chain gene recombination is exhausted 5,6. Regulated expression of RAG-1 and RAG-2 in B cells is thus necessary for both the nearly limitless repertoire of antigen receptors as well as the 'pruning' of this repertoire to maintain central tolerance. Both the pre-BCR and BCR form signaling complexes that suppress Rag transcription at critical stages of B cell development 4,7,8. This negative-feedback regulation of RAG activity by the products of recombination prevents genomic instability in large cycling pre-B cells, contributes to allelic exclusion of heavy-and light-chain expression and inactivates variable-(diversity)
Signaling pathways regulating RAG expression in B lymphocytes
Autoimmunity Reviews, 2009
Development of B-cell lymphopoiesis is dependent on the presence of recombination activating genes RAG1 and RAG2 enzymes. They control the rearrangements of immunoglobulin variable, diversity and joining region segments, and allow progression of the cellular maturation. RAG1 and RAG2 are successively up-and down-regulated at each B-cell stage to progressively generate a B-cell receptor for which unforeseeable antigenic specificity results from a stochastic process. Therefore, in autoreactive immature B cells, new round of RAG re-expression can be observed to eliminate self-reactivity. In some circumstances, RAG up-regulation can also be found in peripheral mature B lymphocytes, specifically in autoimmune diseases. It is therefore of utmost importance to unravel signaling pathways that trigger RAG induction in normal and pathological conditions. Therapeutic modulation of cytokines or intracellular contacts involved in RAG expression might restrict the development of inappropriate autoimmune repertoire.
Immunity, 1995
Two waves of immunoglobulin gene rearrangements, first of the heavy, then of the light chain gene loci form functional immunoglobulin genes during B cell development. In mouse bone marrow the differential surface expression of 8220 (CD45R), c-kit, CD25 and surrogate light chain as well as the cell cycle status allows FACS separation of the cells in which these two waves of rearrangements occur. The gene products of two recombination activating genes, RAG7 and RAG2 are crucial for this rearrangement process. Here, we show that the expression of the RAG genes is twice up-and down-regulated, at the transcriptional level for RAG7 and RAGP, and at the postranscriptional level for RAG2 protein. Expression levels are high in D-, JH and VH-+ DJH rearranging proB and preB-I cells, low in preB cells expressing the preB cell receptor on the cell surface, and high again in VL-, JL rearranging small preB-II cells. In immature B cells expressing on the cell surface RAG1 and RAG2 mRNA is down-regulated, whereas RAG2 protein levels are maintained. Downregulation of RAG7 and RAG2 gene expression after productive rearrangement at one heavy chain allele might be part of the mechanisms that prevent further rearrangements at the other allele.
Molecular and Cellular Biology, 2000
The RAG-2 gene encodes a component of the V(D)J recombinase which is essential for the assembly of antigen receptor genes in B and T lymphocytes. Previously, we reported that the transcription factor BSAP (PAX-5) regulates the murine RAG-2 promoter in B-cell lines. A partially overlapping but distinct region of the proximal RAG-2 promoter was also identified as an important element for promoter activity in T cells; however, the responsible factor was unknown. In this report, we present data demonstrating that c-Myb binds to a Myb consensus site within the proximal promoter and is critical for its activity in T-lineage cells. We show that c-Myb can transactivate a RAG-2 promoter-reporter construct in cotransfection assays and that this transactivation depends on the proximal promoter Myb consensus site. By using a chromatin immunoprecipitation (ChIP) strategy, fractionation of chromatin with anti-c-Myb antibody specifically enriched endogenous RAG-2 promoter DNA sequences. DNase I genomic footprinting revealed that the c-Myb site is occupied in a tissue-specific fashion in vivo. Furthermore, an integrated RAG-2 promoter construct with mutations at the c-Myb site was not enriched in the ChIP assay, while a wild-type integrated promoter construct was enriched. Finally, this lack of binding of c-Myb to a chromosomally integrated mutant RAG-2 promoter construct in vivo was associated with a striking decrease in promoter activity. We conclude that c-Myb regulates the RAG-2 promoter in T cells by binding to this consensus c-Myb binding site. 9203 moters are identical between nucleotides Ϫ70 and Ϫ50. Within this region, we identified a binding site for the B-cell-specific transcription factor BSAP (Pax-5) and showed that BSAP binds this sequence both in vitro and in vivo. Furthermore, mutations which disrupt BSAP binding greatly diminish B-cellspecific promoter activity. T cells, however, do not express BSAP but still require sequences in the conserved Ϫ70 to Ϫ50 promoter region for RAG-2 promoter activity (32). Recent reports suggested that c-Myb, a hematopoietic lineage-restricted transcription factor, is preferentially expressed in developing T but not B cells (2, 11) (see below). In the experiments described below, we have gone on to determine that c-Myb binds to the RAG-2 promoter and is critical for RAG-2 promoter activity in T cells.
Distinct functions for the transcription factor Foxo1 at various stages of B cell differentiation
2008
The Foxo transcription factors (Foxo1, Foxo3, Foxo4) modulate cell fate decisions in diverse systems. Here we show that Foxo1-dependent gene expression was critical at multiple stages of B cell differentiation. Early deletion of Foxo1 caused a severe block at the pro-B cell stage, due to a failure to express interleukin 7 receptor α (IL-7Rα). Foxo1 inactivation in late pro-B cells resulted in an arrest at the pre-B cell stage due to a reduction in Rag1 and Rag2 expression. Deletion of Foxo1 in peripheral B cells led to fewer lymph node B cells due to reduced L-selectin expression, and failed class switch recombination due to impaired Aicda upregulation. Thus, Foxo1 regulates a transcriptional program that is essential for early B cell development and peripheral B cell function. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling
Journal of immunology (Baltimore, Md. : 1950), 2016
The recombination activating gene (RAG) 1 and RAG2 protein complex introduces DNA breaks at Tcr and Ig gene segments that are required for V(D)J recombination in developing lymphocytes. Proper regulation of RAG1/2 expression safeguards the ordered assembly of Ag receptors and the development of lymphocytes, while minimizing the risk for collateral damage. The ataxia telangiectasia mutated (ATM) kinase is involved in the repair of RAG1/2-mediated DNA breaks and prevents their propagation. The simultaneous occurrence of RAG1/2-dependent and -independent DNA breaks in developing lymphocytes exposed to genotoxic stress increases the risk for aberrant recombinations. In this study, we assessed the effect of genotoxic stress on RAG1/2 expression in pre-B cells and show that activation of the DNA damage response resulted in the rapid ATM-dependent downregulation of RAG1/2 mRNA and protein expression. We show that DNA damage led to the loss of FOXO1 binding to the enhancer region of the RAG...
Blood, 2015
In developing lymphocytes, expression and activity of the RAG1 and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes, and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALL), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1-phase of the cell-cycle. In this study we show a novel pathway that suppresses RAG expression in cycling transformed mouse pre-B cells and human pre-B B-ALL cells, involving the negative regulation of FOXO1 by NF-κB. Inhibition of NF-κB in cycling pre-B cells resulted in up-regulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2 and Td...
Molecular and Cellular Biology, 2012
Immunoglobulin (Ig) genes naturally acquire frequent premature termination codons during the error-prone V(D)J recombination process. Although B cell differentiation is linked to the expression of productive Ig alleles, the transcriptional status of nonfunctionally recombined alleles remains unclear. Here, we tracked transcription and posttranscriptional regulation for both Ig heavy-chain (IgH) alleles in mice carrying a nonfunctional knock-in allele. We show that productively and nonproductively VDJ-rearranged alleles are transcribed throughout B cell development, carry similar active chromatin marks, and even display equivalent RNA polymerase II (RNAPII) loading after B cell stimulation. Hence, these results challenge the idea that the repositioning of one allele to heterochromatin could promote the silencing of nonproductive alleles. Interestingly, the efficiency of downstream RNA surveillance mechanisms fluctuates according to B cell activation and terminal differentiation: unspliced nonfunctional transcripts accumulate in primary B cells, while B cell activation promotes IgH transcription, RNA splicing, and nonsense-mediated mRNA decay (NMD). Altogether, IgH transcription and RNA splicing rates determine by which RNA surveillance mechanisms a B cell can get rid of nonproductive IgH mRNAs.