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. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5' of the B cell-specific Erag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1. Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase.

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. We show that RAG-induced DNA double-strand breaks (DSBs) generated during IgL chain gene assembly paradoxically promote pre-B cell survival. This occurs through the ATM-dependent induction of Pim2 kinase expression. Similar to Pim1, Pim2 phosphorylates BAD, which antagonizes the pro-apoptotic function of BAX. However, unlike IL-7 induction of Pim1, RAG DSB-mediated induction of Pim2 does not drive proliferation. Rather, Pim2 has antiproliferative functions that prevent the transit of pre-B cells harboring RAG DSBs from G1 into S phase, where these DNA breaks could be aberrantly repaired. Thus, signals from IL-7 and RAG DSBs activate distinct Pim kinase family members that have context-dependent activities in regulating pre-B cell proliferation and survival.

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. We found that inhibition of v-Abl activity results in upregulation of transcription of the pro-apoptotic TNF-family ligand tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL). Similarly to BCR-Abl-transformed human cells, activation of the transcription factor Foxo3a led to increased TRAIL transcription and induction of a G1 arrest in the absence of v-Abl inhibition, and this effect could be inhibited by the expression of a constitutively active AKT mutant. Multiple pathways act to inhibit FoxO3a activity within Abelson cells. In addition to diminishing transcription factor activity via inhibitory phosphorylation by AKT family members, we found that inhibition of IKKbeta activity results in an increase in the total protein level of FoxO3a. Furthermore overexpression of the p65 subunit of NF-kappaB results in an increase in TRAIL transcription and in apoptosis and deletion of IKKalpha and beta diminishes TRAIL expression and induction. We conclude that in Abelson cells, the inhibition of both NF-kappaB and FoxO3a activity is required for suppression of TRAIL transcription and maintenance of the transformed state.

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. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

Biallelic, ubiquitous transcription from the distal germline Ig{kappa} locus promoter during B cell development.

Allelic exclusion of Ig gene expression is necessary to limit the number of functional receptors to one per B cell. The mechanism underlying allelic exclusion is unknown. Because germline transcription of Ig and TCR loci is tightly correlated with rearrangement, we created two novel knock-in mice that report transcriptional activity of the Jkappa germline promoters in the Igkappa locus. Analysis of these mice revealed that germline transcription is biallelic and occurs in all pre-B cells. Moreover, we found that the two germline promoters in this region are not equivalent but that the distal promoter accounts for the vast majority of observed germline transcript in pre-B cells while the activity of the proximal promoter increases later in development. Allelic exclusion of the Igkappa locus thus occurs at the level of rearrangement, but not germline transcription.

Foxo1 directly regulates the transcription of recombination-activating genes during B cell development.

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.

NF-kappa B comes home.

NF-kappa B was discovered because of its binding to the Ig kappa locus intronic enhancer, but deletion of its binding site does not appear to affect V-to-J kappa rearrangement. New work by Verkoczy et al. in this issue of Immunity suggests that NF-kappa B regulates Ig kappa rearrangement after all, by activating RAG expression during receptor editing.