Foxo1 and Foxo3a are the predominantly expressed family members in B cells (data not shown)

Foxo1 and Foxo3a are the predominantly expressed family members in B cells (data not shown). To evaluate whether Foxo transcription factors are required for GADD45a to activate transcription of the locus, we expressed shRNA AGN 196996 molecules targeting and individually in AMuLV-transformed substantially diminished GADD45a-induced GFP expression, whereas knockdown of or had little effect (Fig. 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 heavy-chain locus rearrangement and then again at the pre-B cell stage during immunoglobulin AGN 196996 light-chain locus rearrangement2,3. Productive rearrangement of both heavy- and light-chain genes leads to BCR expression at the immature B cell stage. Basal AGN 196996 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 (and expression continues, resulting in further light-chain locus rearrangement (receptor editing) and altered BCR specificity until an innocuous BCR is usually expressed or the potential for light-chain gene Prkg1 recombination is usually exhausted5,6. Regulated expression of RAG-1 and RAG-2 in B cells is usually 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 transcription at critical stages of B cell development4,7,8. 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)-joining recombination to stabilize genes encoding a self-tolerant receptor. The signaling pathways and transcription factors that mediate this regulation are ill defined. Given this lack of knowledge, we devised a functional screen for cDNA molecules able to induce transcription in a transformed pro-B cell line. We found that the stress-regulated protein GADD45a (A001020) activated transcription in these cells by a pathway involving mitogen-activated protein kinase signaling and the transcription factor Foxo1 (A000944). We also found that phosphatidylinositol-3-OH kinase (PI(3)K), the serinethreonine kinase Akt and Foxo1 were important in regulating transcription in developing primary bone marrow B cells and during receptor editing. RESULTS Screen for regulators of transcription identifies GADD45a To screen for factors that regulate transcription in B lymphocytes, we generated an indicator cell line using a published knock-in mouse9 in which the endogenous coding sequence is replaced with cDNA encoding green fluorescent protein (and that GFP expression was an accurate reflection of promoter activity (data not shown). We then infected bone marrow from selectively transforms cells and arrests their development at a stage that resembles that of large cycling pre-B cells10. Treatment of AMuLV-transformed pro-B cells with a small-molecule inhibitor of v-Abl, STI-571 (Gleevec), results in an increase in transcription of genes common of pre-B cells, including and (ref. 11). As expected, treatment with STI-571 induced GFP expression in these AMuLV-transformed by a retroviral cDNA library screen for factors that induce transcription in AMuLV-transformed B cells. (a) Flow cytometry of GFP expression in AMuLVtransformed (GADD45a), labeled with anti-Thy-1.1 (retroviral marker) and gated for infected Thy-1.1+ cells (solid line) or uninfected Thy-1.1? cells (filled histogram). Numbers above bracketed lines indicate percent GFP+ cells in the infected population (top number) and uninfected population (bottom number in parenthesis). (c) Quantitative RT-PCR analysis of and transcripts in sorted cells infected with empty vector retrovirus or retrovirus expressing GADD45a. Values are normalized to transcript abundance and are presented relative to expression in cells transduced with empty vector, set as 1. All data are representative of at least three impartial experiments. We infected AMuLV-transformed and transcripts from the unaltered allelic locus in sorted cells overexpressing GADD45a (Fig. 1c). Characterization of the GADD45a pathway was initially identified as a gene induced by DNA damage in Chinese hamster ovary cells12. The protein it encodes is usually one of three related proteins, GADD45a, GADD45b and GADD45g, that share over 50% amino acid identity. All three are induced by various cell stresses, including DNA damage, hypoxia and withdrawal of growth factor13. Among their other known functions, GADD45 proteins bind to and activate the mitogen-activated protein kinase MEKK4, which in turn leads to phosphorylation of the stress-associated protein kinases p38 and Jnk14. In T cells, GADD45 proteins are involved in propagating signals from the TCR or cytokine receptors to p38 and Jnk15-17. Additionally, and have been identified as transcripts induced in AMuLV-transformed pro-B cells treated with the Abl kinase inhibitor STI-571 (ref. 11). To test whether the GADD45a overexpression phenotype was MEKK4 dependent, we created an inducible from of AGN 196996 GADD45a by fusing.