Dual Mechanism of Gene Repression by c-Myb during Pre-B Cell Proliferation.

Developing B lymphocytes undergo clonal expansion following successful immunoglobulin heavy chain gene rearrangement. During this proliferative burst, expression of the genes is transiently repressed to prevent the generation of double-stranded DNA (dsDNA) breaks in cycling large pre-B cells. The genes are then reexpressed in small, resting pre-B cells for immunoglobulin light chain gene rearrangement.

Gfi1 and gfi1b repress rag transcription in plasmacytoid dendritic cells in vitro.

Growth factor independence genes (Gfi1 and Gfi1b) repress recombination activating genes (Rag) transcription in developing B lymphocytes. Because all blood lineages originate from hematopoietic stem cells (HSCs) and different lineage progenitors have been shown to share transcription factor networks prior to cell fate commitment, we hypothesized that GFI family proteins may also play a role in repressing Rag transcription or a global lymphoid transcriptional program in other blood lineages. We tested the level of Rag transcription in various blood cells when Gfi1 and Gfi1b were deleted, and observed an upregulation of Rag expression in plasmacytoid dendritic cells (pDCs).

Ebf1 and c-Myb repress rag transcription downstream of Stat5 during early B cell development.

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.

MK5 activates Rag transcription via Foxo1 in developing B cells.

Foxo1 is a critical, direct regulator of Rag (recombination activating gene) transcription during B cell development and is thus essential for the generation of a diverse repertoire of antigen receptors. Although Foxo1 regulation has been widely studied in many cell types, pathways regulating Foxo1 in B cells have not been fully elucidated. By screening a panel of Foxo1 mutants, we identified serine 215 on Foxo1 as a novel phosphorylation site that is essential for the activation of Rag transcription.

miR290-5p/292-5p activate the immunoglobulin kappa locus in B cell development.

Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer.

Extensive gene-specific translational reprogramming in a model of B cell differentiation and Abl-dependent transformation.

To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells.

RAG-induced DNA double-strand breaks signal through Pim2 to promote pre-B cell survival and limit proliferation.

Interleukin 7 (IL-7) promotes pre-B cell survival and proliferation by activating the Pim1 and Akt kinases. These signals must be attenuated to induce G1 cell cycle arrest and expression of the RAG endonuclease, which are both required for IgL chain gene rearrangement. As lost IL-7 signals would limit pre-B cell survival, how cells survive during IgL chain gene rearrangement remains unclear.

Gfi1b negatively regulates Rag expression directly and via the repression of FoxO1.

Precise regulation of Rag (recombination-activating gene) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus.

CTCF-binding elements mediate control of V(D)J recombination.

Immunoglobulin heavy chain (IgH) variable region exons are assembled from V(H), D and J(H) gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires.

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells.

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus.

Forced expression of cyclin-dependent kinase 6 confers resistance of pro-B acute lymphocytic leukemia to Gleevec treatment.

The gene encoding c-ABL, a nonreceptor protein tyrosine kinase, is involved in a chromosomal translocation resulting in expression of a BCR-Abl fusion protein that causes most chronic myelogenous and some acute lymphocytic leukemias (CML and ALL) in humans. The Abelson murine leukemia virus (A-MuLV) expresses an alternative form of c-Abl, v-Abl, that transforms murine pro-B cells, resulting in acute leukemia and providing an experimental model for human disease. Gleevec (STI571) inhibits the Abl kinase and has shown great utility against CML and ALL in humans, although its usefulness is limited by acquired resistance.

Allelic exclusion of immunoglobulin genes: models and mechanisms.

The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B-cell development, the relevance of monospecificity to B-cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Ig kappa and Ig lambda light chain genes.

Epigenetics drives RAGs to recombination riches.

V(D)J recombination of antigen receptor gene segments in B and T cells is mediated by the lymphoid-specific proteins RAG1 and RAG2. Now, Ji et al. (2010) demonstrate how RAG1 and RAG2 use DNA sequence specificity and modified histones within chromatin to target specific loci for V(D)J recombination at different stages of lymphoid development.

Abelson virus transformation prevents TRAIL expression by inhibiting FoxO3a and NF-kappaB.

The Abelson Murine Leukemia Virus (A-MuLV) encodes v-Abl, an oncogenic form of the ubiquitous cellular non-receptor tyrosine kinase, c-Abl. A-MuLV specifically transforms murine B cell precursors both in vivo and in vitro. Inhibition of v-Abl by addition of the small molecule inhibitor STI-571 causes these cells to arrest in the G1 phase of the cell cycle prior to undergoing apoptosis.

NF-kappaB activity marks cells engaged in receptor editing.

Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity.

A histone code for regulating V(D)J recombination.

In a recent issue of Molecular Cell, Shimazaki et al. (2009) show that an interaction between RAG2 and a methylated histone might play a critical regulatory role in V(D)J recombination by enhancing DNA binding and enzymatic activity of the V(D)J recombinase.

Development of immunoglobulin lambda-chain-positive B cells, but not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals.

By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain.

Mechanisms controlling expression of the RAG locus during lymphocyte development.

Recombination activating genes (RAG)1 and RAG2 are expressed in developing B and T lymphocytes and are required for the rearrangement of antigen receptor genes. In turn, RAG expression is regulated by the products of these assembled immunoglobulin (Ig) and T cell receptor (TCR) genes. Upon successful assembly of Ig genes, the antigen receptor is expressed on the immature B cell surface and tested for autoreactivity leading to either maintenance or inactivation of RAG expression.

The effects of c-Abl mutation on developing B cell differentiation and survival.

c-Abl is a widely expressed Src family protein tyrosine kinase that is activated by chromosomal translocation in certain human leukemias. While shown in various experimental systems to regulate cell division and stress responses, its biological functions remain poorly understood. Although expressed at similar levels throughout B cell development, we found that the fraction of phosphorylated, active c-Abl peaks at the pro-B stage.