HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, J. Articles by Fruman, D. A. Search for Related Content PubMed PubMed Citation Articles by Chen, J. Articles by Fruman, D. A.

FOXO Transcription Factors Cooperate with EF1 to Activate Growth Suppressive Genes in B Lymphocytes¹

Jing Chen^*, Isharat Yusuf^*, Hilde-Marie Andersen^* and David A. Fruman^2,

^* Department of Molecular Biology and Biochemistry, and Center for Immunology, University of California, Irvine, CA 92697

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Forkhead transcription factors regulate many aspects of lymphocyte development and function. The FOXO subgroup of Forkhead factors opposes proliferation and survival, and FOXO inactivation is an important outcome of Ag receptor signaling. FOXO activity at target promoters is modulated by other transcription factors in a manner dependent on cell type and external stimulus. We have investigated the mechanisms by which FOXO proteins activate the promoters of two target genes in murine B lymphocytes, Ccng2 (encoding cyclin G₂) and Rbl2 (p130), each of which has been implicated in cell cycle arrest. FOXO proteins bound directly to both promoters in vitro and in vivo, augmented transcriptional activity in reporter assays, and increased expression of the endogenous genes. Each of the promoter sequences has consensus binding sites for the EF1 transcription factor, previously shown to either repress or activate different promoters. EF1 bound to the Ccng2 and Rbl2 promoters in vitro and in vivo and increased reporter activity as well as endogenous mRNA levels for these genes. Strikingly, EF1 synergized with FOXO proteins to strongly activate transcription from both promoters. Coexpression of EF1 enhanced FOXO-induced cell cycle arrest in B lymphoma cells. These findings establish a novel mechanism of FOXO function at target promoters: cooperation with EF1.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The FOXO transcription factors play an evolutionarily conserved role in the control of metabolism, proliferation, survival, stress resistance, and longevity (1, 2, 3). Four FOXO family members exist in mammals: FOXO1, FOXO3a, FOXO4, and FOXO6. Although gene disruption experiments are beginning to reveal that these isoforms have unique functions (4, 5, 6), some general aspects of FOXO regulation and function appear similar among family members. Expression of FOXO proteins induces cell cycle arrest in many cell types (1, 2, 3). In growth factor-dependent hemopoietic cell lines, increased FOXO function is also accompanied by decreased survival (7, 8, 9). In contrast, FOXO expression in fibroblasts can promote cellular quiescence (10, 11). Growth factor signaling leads to the inactivation of endogenous FOXO factors, thus allowing survival and cell cycle progression. FOXO inactivation is accomplished in part by phosphorylation mediated by Akt, a serine/threonine kinase activated downstream of PI3K (2, 3). Remarkably, the pathway leading from growth factor receptors to PI3K and Akt, and thence to FOXO phosphorylation and inactivation, is conserved from Caenorhabditis elegans to humans.

Decreased metabolic activity and resistance to stress and apoptosis are hallmarks of the quiescent state, also known as G₀ phase of the cell cycle (12). Mature lymphocytes are maintained in this quiescent state until recognition of Ag. Several observations suggest that FOXO transcription factors help to maintain lymphocyte quiescence. Activation of PI3K is a central and required feature of lymphocyte cell cycle entry (13, 14). FOXO1 and FOXO3a are expressed in resting T and B cells, and rapidly phosphorylated and deactivated in a PI3K-dependent manner following ligation of Ag receptors (9, 15). Microarray analysis has revealed a cluster of genes down-regulated in a PI3K-dependent manner following B cell activation, consistent with possible control by FOXO factors (16). In addition, deletion of the FOXO3a gene in mice results in a reduced threshold for T cell activation and the development of spontaneous autoimmunity (6).

Given the importance of FOXO transcription factors in cell metabolism, proliferation, survival, and other processes, considerable effort has been directed at identifying FOXO target genes. FOXO factors can bind directly to a consensus DNA element (TTGTTTAC) termed the insulin response sequence (IRS).³ This interaction was defined initially in the promoter for insulin-like growth factor-binding protein-1, and the consensus sequence was established later by affinity selection experiments (17). Sequences related to this motif have been found in the promoters of a number of genes that appear to be regulated by FOXO factors. In addition, transcriptional profiling has provided a global view of FOXO-dependent gene regulation in C. elegans and in human tumor cells (18, 19). It is becoming apparent that the spectrum of FOXO target genes differs depending on cell type and external stimuli, which affects the array of partner transcription factors as well as coactivators and corepressors at target promoters (20). Interestingly, several functionally important FOXO target genes in tumor cells are not dependent on FOXO binding to an IRS element (19), suggesting that FOXO proteins can regulate transcription indirectly through partner proteins or directly via nonconsensus-binding motifs.

Little is known about FOXO target genes that control proliferation of mature lymphocytes. p27^Kip and Bim were reported to be up-regulated in IL-2-dependent T cells transfected with FOXO3a (9). We found that activation of mouse B cells is accompanied by the PI3K-dependent down-regulation of Ccng2 (encoding the protein cyclin G₂) and Rbl2 (encoding the retinoblastoma-like protein p130/Rb2) (15), two genes implicated in FOXO-dependent quiescence in fibroblasts (11, 21). In this study, we show that FOXO proteins bind to consensus IRS elements in each of these promoters in vitro and are associated with the promoters in primary splenic B cells. FOXO factors activate transcription from the murine Ccng2 and Rbl2 promoters in B lymphoma cells, and augment mRNA levels of the endogenous genes. We also demonstrate that the zinc finger transcription factor EF1 binds to each of these promoters and increases endogenous gene expression. In reporter assays, EF1 activates transcription on its own and synergizes strongly with FOXO proteins. Interestingly, FOXO-dependent activation of these promoters and synergy with EF1 do not require canonical FOXO-binding DNA elements. Finally, coexpression of EF1 enhances cell cycle arrest and target gene expression induced by FOXO proteins in B lymphoma cells. These findings establish a novel functional cooperation in B cells between FOXO transcription factors and EF1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Primary cells and cell lines

BALB/c mice were purchased from The Jackson Laboratory and bred in our colony. Animals were housed and studied in accordance with protocols approved by the Institutional Animal Care and Use Committee. Splenic B cells were purified, as previously described (15).

A20 (from American Type Culture Collection) and M12 (from M. Schlissel, University of California, Berkeley, CA) B lymphoma cells were maintained in RPMI 1640 medium supplemented with 10% FCS (HyClone), 10 mM HEPES, 2 mM L-glutamine, 50 µM 2-ME (Sigma-Aldrich), and antibiotics in a humidified incubator at 37°C in 5% CO₂. During experiments, the cultures were maintained in the exponential phase of growth. All supplements not indicated were obtained from Mediatech.

Cos7 cells were obtained from C. Hughes (University of California, Irvine, CA). The 293T cells were obtained from American Type Culture Collection. Both cell lines were maintained in DMEM supplemented with 10% FBS and 2 mM glutamine.

Reporter and expression plasmids

A 1464-bp fragment corresponding to the Rbl2 5' flanking sequence –1413 to +51 was amplified by PCR from the mouse genomic DNA using the primer set CGC GGA TCC TGC TCC CGC AAC ACA TGT GAG and CGC GGA TCC GCG GCT CTT CAG ATG CGC TCA GG with a BamHI site at both of the primer ends. A 2200-bp fragment corresponding to the Ccng2 5' flanking sequence –1554 to +646 was amplified by PCR from the mouse genomic DNA using the primer set GGG GTA CCC CAG TCT TCT TGT TCG GCT CC and GGG GTA CCC CTG TCA GTC CCT CAG CTG CG with a KpnI site at both of the primer ends. Both PCR fragments were gel purified and cloned into the pDrive cloning vector (Qiagen). The Rbl2 promoter fragment was excised with BamHI and subcloned into the BglII site of pGL2.Basic (Promega). The Ccng2 promoter fragment was excised with KpnI and subcloned into the same site of pGL2.Basic. Sequence and orientation were checked both by restriction enzyme digestion and sequencing (University of California Davis sequencing). A series of pGL2.Rbl2 promoter and pGL2.Ccng2 truncation mutants, designed to remove potential FOXO and EF1 DNA binding sites, were created by PCR using a series of nested 5' and 3' oligonucleotide primers. Those promoters designed to mutate the putative binding site FOXO and EF1 DNA binding sites were created by oligonucleotide-mediated mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene) and subcloned into the MCS site of pGL2.Basic. The DNA sequence of each promoter mutant was confirmed before use in transient transfection assays.

pGL2.TKmin promoter reporter was provided by M. Waterman (University of California, Irvine, CA). pGL2.PU.1 was provided by E. Rothenberg (California Institute of Technology, Pasadena, CA). The pMIT empty vector, pMIT.FOXO1, pMIT.FOXO1.A3, pMIT.FOXO3a, and pMIT.FOXO3a-A3 vectors used for expression of FOXO genes were previously described (15). pECE.FOXO3a-A3-ER was provided by A. Brunet (Stanford University, Stanford, CA). To subclone into the pMIT vector, pECE.FOXO3a-A3-ER was digested with BglII (partial) and NotI to obtain the 5' 2-kb fragment, and inserted into the same sites of the pMIT vector. pECE.FOXO3a-A3-ER was then digested with NotI to obtain the 3' 1-kb fragment and ligated with the 2-kb fragment in pMIT at NotI site. The sequence and orientation were checked both by restriction enzyme digestions and sequencing.

pcDNA3.1/mEF1 was a gift from Y. Higashi (Osaka University, Osaka, Japan).

pCI.CycG2 vector encoding the cyclin G₂ cDNA was obtained from E. Conner (National Cancer Institute, Bethesda, MD). pCI.CycG2 was digested by XhoI and NotI, and the insert was cloned into same sites of the pMSCV.IRES.hCD4 (pMIC) vector. pcDNA3.Rbl2/p130 encoding the human Rbl2/p130 cDNA was a gift from A. Giordano (Temple University, Philadelphia, PA) and was digested with BamHI and NotI and cloned into the BglII and NotI sites of pMIT.

Transient transfection experiments and luciferase assays

Transient transfection experiments in A20 or M12 cells were performed by electroporation (Bio-Rad Genepulser). Exponentially growing cells were washed with RPMI 1640 medium without serum and resuspended in RPMI 1640 medium at 2 x 10⁷ cells/ml. A total of 500 µl of cell suspensions was mixed with the promoter-reporters and various expression vectors and incubated for 10 min at room temperature. Cells and DNA mixture were then transferred to 4-mm Genepulser cuvettes (Invitrogen Life Technologies) and electroporated at 300 V and 960 µF. The total amount of DNA per transfection was made equal by the addition of an appropriate amount of the pGEM3Z vector (Promega) or pMIT or pcDNA3.1 (Invitrogen Life Technologies) empty vectors. After electroporation, cells were immediately transferred to tissue culture flask with 10 ml B cell medium. Forty hours posttransfection, the cells were lysed and luciferase activity was measured using a commercial luciferase assay kit (Promega). Luciferase activity was normalized based on protein concentration, as assayed by BCA Protein Assay Reagent (Pierce).

EMSA

Cos7 cells were transfected with 10 µg of pECE.Foxo3a-A3, pcDNA3.FOXO1, pcDNA3.FOXO1H215R, pcDNA3.FOXO1.A3, or pcDNA3.1 empty vector by calcium phosphate precipitation. At 48 h posttransfection, cells were washed twice with PBS, and 300 µl of lysis buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 µM ZnCl₂, 100 µM sodium orthovanadate, 1% Triton X-100, and protease inhibitors (Complete, protease inhibitor mixture tablet; Roche)) was added to each dish. Cells were harvested by scraping and transferred into microcentrifuge tubes, vortexed for 30 s, and centrifuged at the maximum setting for 20 min at 4°C. Supernatants were aliquoted to microcentrifuge tubes and stored at –80°C. The 293T cells were transfected by pcDNA3.EF1myc or pcDNA3.1, as described for Cos7 cells. EMSA was conducted using Cos7 or 293T cell extracts, as described (22). A total of 100 ng of a double-stranded oligodeoxynucleotide, containing various FOXO or EF1 binding sites, was labeled with [-³²P]dCTP (3000 mCi/mmol; PerkinElmer) by fill-in reaction using the Klenow fragment of DNA polymerase I. Unincorporated nucleotides were removed from the labeling reactions using the Nucleotide Removal Kit (Qiagen). For supershift experiments, nuclear extracts were preincubated with 1 µg of anti-FOXO3a (Upstate Biotechnology) and anti-human c-Myc epitope 9E10 (Santa Cruz Biotechnology) Abs for 30 min at 4°C. Competition experiments were performed with 1:100 molar ratios of labeled to nonlabeled oligonucleotides. The oligonucleotides used for labeling were as follows: Rbl2-foxo-a (5'-CTA GTT TAT TTT GTT TTT GTT TG-3'), Rbl2-foxo-b (5'-AGC TCG TTT TTT GCT TTC TTC TTC-3'), Ccng2-foxo-a (5'-AGA AAG TAA AAC AAA CAA A CA AAA CCA AAAC-3'), Ccng2-foxo-b (5'-CTG AAA ACC AAA AGC AAA CAG TAC AAC-3'), Ccng2-foxo-c (5'-CTA GAC AGC GAA AAC AAA ACA AAT CGG-3'), Ccng2-foxo-d (5'-ATC GCA GAC TCA AAAC AAA AAC AAG G-3'), Ccng2-EF1-a (5'-GAC CTT TAA AAG GTG TGA GGC GT-3'), and Rbl2-EF1-c (5'-CGC ACT CTC CTC CCT CAG GTG GCT CAG-3'). The sequence of mutant oligos used for cold competitions are: Rbl2-foxo-a-mut (5'-CTA GTT TCT TTT CTT TTT CTT TC-3'), Ccng2-foxo-a-mut (5'-AGA AAG TAA AAG AAA GAA A GA AAA GCA AAAC-3'), and Ccng2-foxo-d-mut (5'-ATC GCA GAC TCA AAAG AAA AAG AAG G-3').

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed using the anti-acetylated histone H3 Ab-based ChIP assay kit (Upstate Biotechnology) with minor modifications. In brief, 37% formaldehyde solution was added directly to purified primary B cells at a final concentration of 1% and cells were incubated for 10 min at 37°C. Cells were harvested and washed twice with ice-cold PBS containing protease inhibitors (Roche) and lysed with SDS lysis buffer for 10 min on ice. The lysate was sonicated to an average length of 500-1000 bp and clarified by centrifuging at 16,000 x g for 10 min at 4°C. The samples were then precleared with salmon-sperm DNA-protein A-Ag beads (Upstate Biotechnology) for 2 h. After a brief spin, the supernatant fraction was collected and diluted 10-fold with ChIP dilution buffer and split into five equal parts for immunoprecipitation with either normal rabbit IgG (Santa Cruz Biotechnology; SC-2027), anti-FOXO1 (Cell Signaling Technology), anti-FOXO3a (Upstate Biotechnology), anti-EF1 (anti-ZEB1; Santa Cruz Biotechnology), or no Ab. Immunoprecipitation was done by overnight rotation at 4°C. Beads were harvested, washed, and eluted per the manufacturers’ instructions. Cross-links were reversed by heating at 65°C for 4 h. DNA was purified by phenol-chloroform extraction and recovered by ethanol precipitation. Precipitated DNA was analyzed by PCR amplification for 25 cycles (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min), separated on 1.5% Agarose gel, transferred to Nylon membranes, and hybridized to each probe (same as the ChIP PCR fragments). The primer sets used for PCR are as follows: Rbl2 forward, 5'-GTG TGA TTG AGG GTT GAG GGT TGA G-3'; Rbl2 reverse, 5'-GCC TGC TGG GAA ATG TAG TCT CG-3'; Ccng2 forward, 5'-CAG AGG ACG CAA CAG CCA GTC GG-3'; Ccng2 reverse, 5'-CCG CAG CCT CCG GAG CTG AGC-3'. Probes were amplified by PCR from the cloned promoters using the same primer sets. The PCR products were gel purified using the Qiagen gel extraction kit and radiolabeled by using RadPrime DNA Labeling System (Invitrogen Life Technologies). Hybridization was performed at 42°C for 24 h, according to the protocol provided by BD Clontech. Filters were exposed to a phosphor screen and visualized by using a phosphor imager system. For the ChIP control, PCR primers for Carbonic anhydrase I (CA-I) promoter were provided by T. Bender (University of Virginia, Charlottesville, VA). The CA-I ChIP DNA was PCR amplified for 35 cycles (94 °C for 1 min, 58°C for 45 s, 72°C for 45 s, with a 7-min final extension at 72°C), and PCR samples were then resolved on 2.5% agarose gels and visualized by ethidium bromide staining.

Northern blot and Q-PCR analysis

A20 cells were transfected by electroporation, as described above. Five batches of 10⁷ cells received 10 µg of pMIT-FOXO3a.A3-ER and 10 µg of either empty vector (pMIT) or pMIT-EF1. Forty-eight hours after transfection, cells were harvested, pooled, and resuspended in 40 µl of MACS buffer per 10⁷ total cells. Cells were first stained with anti-Thy-1.1-biotin (eBioscience) and then incubated with anti-biotin microbeads (Miltenyi Biotec), and purified via positive selection on MACS columns per the manufacturers’ instructions. Purity of Thy-1.1-positive cells was greater than 70% via FACS analysis. Cells were cultured for an additional 16 h in the absence or presence of 400 nM 4-hydroxytamoxifen (Sigma-Aldrich). Total cellular RNA was extracted by using TRIzol reagent (Invitrogen Life Technologies). Fractionation of RNA and transfer to nitrocellulose membranes were conducted, as previously described (23). The following [-³²P]dCTP probes were used: a NotI and XhoI fragment encoding the whole mouse Ccng2 cDNA cut from the pCI.CycG2, a 578-bp mouse Rbl2 cDNA fragment amplified from A20 cells by RT-PCR (forward primer, 5'-CCA GTG ATG AGG TCA AAC AGC ACC-3'; reverse primer, 5'-GCA TTT GCT GCC ACA CTG AGT CAG-3'), and a 1.2-kb PstI fragment of chicken GAPDH cDNA fragment (23). Hybridization conditions were conducted, as described for the ChIP assays. Filters were exposed to a phosphor screen, and results were quantified using ImageQuant software (Amersham). cDNA was synthesized, and real-time quantitative PCR using SyBr Green was performed, as described, using primers specific for p130, cyclin G₂, and -actin cDNA (15).

Retroviral infection and cell cycle analysis of primary B cell blasts were performed, as described (15). Cell cycle analysis of transfected A20 cells was done similarly. In both cases, FlowJo software was used to calculate cell cycle distribution of cell populations gated by expression of the IRES-linked surface marker gene product.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cyclin G₂ suppresses B cell proliferation, and the Ccng2 promoter contains IRS motifs

Previously, we reported that retroviral transduction of FOXO proteins into activated splenic B cell blasts induces partial cell cycle arrest or delay (15). We also noted that mRNA levels of Ccng2 and Rbl2 are diminished following BCR cross-linking of splenic B cells, in a manner partially dependent on PI3K (15). These observations suggested that Ccng2 and Rbl2 might be FOXO target genes in B cells. Both cyclin G₂ and p130/Rb2 can suppress cell proliferation when overexpressed in certain cell types (24, 25). To determine whether expression of cyclin G₂ or p130 alone has any effect on cell cycle progression in this system, we generated retroviruses containing cDNAs for these proteins. Retroviral transduction of cyclin G₂, but not p130, caused an increase in the fraction of B cells in G₀-G₁ phase of the cell cycle, with a concominant decrease in S phase (Fig. 1). The effect was comparable to that achieved by expression of wild-type FOXO1 or FOXO3a. These findings are consistent with the possibility that induction of cyclin G₂ by FOXO expression contributes to cell cycle arrest in activated mature B cells. As observed previously (15), A3 mutants of FOXO proteins in which all Akt phosphorylation sites have been mutated were more effective at inducing cell cycle arrest/delay, with FOXO1.A3 also causing a significant decrease in G₂-M phase.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Cyclin G2 induces cell cycle arrest or delay in primary B cell blasts. LPS-stimulated splenic B cells were infected with MSCV-based retroviruses expressing cyclin G2, p130, FOXO1, FOXO1.A3, FOXO3a, FOXo3a.A3, or their empty vector controls (pMIC or pMIT). Cell cycle analysis was performed 48 h later using propidium iodide, gating on marker-positive (hCD4 or Thy1.1) cells. The data represent the mean ± SEM of three independent experiments. A t test was done to determine statistical differences of cell cycle stages relative to empty vector control (pMIC or pMIT). *, p < 0.05; **, p < 0.005.

View larger version (65K): [in this window] [in a new window]   FIGURE 2. Locations of consensus binding sites (underlined) for FOXO and EF1 transcription factors in the regulatory regions of the Ccng2 and Rbl2 genes. The start sites of transcription (obtained from the Ensembl Mouse Genome Server) are shown as nucleotide no. 1. Bold type, primers used to clone the promoters; italic type, primers used to clone del5 fragment.

We tested the ability of FOXO proteins to activate transcription from these promoters in reporter assays. Fragments of the Ccng2 and Rbl2 promoters were cloned upstream of the luciferase gene in pGL2.Basic. Transient transfections were conducted in A20 B lymphoma cells. Both promoter-reporters were more basally active in A20 cells compared with the pGL2.Basic vector or the promoters cloned in the reverse orientation (Fig. 3, A and B). Cotransfection of FOXO1 or FOXO3a enhanced this activation in a dose-dependent manner, ranging from 1.5- to 10-fold over promoter-reporter alone (Fig. 3, A and B).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Transfection of A20 cells with FOXO constructs augments transcription of reporter genes driven by the Ccng2 or Rbl2 promoters. A and B, A20 cells were electroporated with 5 µg of pGL2.Basic, pGL2.Ccng2(–) or pGL2.Ccng2(+) (A) and pGL2.Rbl2(–) or pGL2.Rbl2(+) (B) alone or with FOXO expression vectors as indicated. The empty vector pMIT was added to make the total amount of DNA equal in all transfections. The bars indicate the average luciferase activity normalized to protein concentration. Luciferase activity of the reporter gene in the absence of exogenous transactivator was designated as one. All luciferase data shown represent the average of three independent experiments. Error bars indicate SD of the mean.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Akt opposes promoter activation by wild-type FOXO. cDNAs for wild-type Akt or kinase-dead (K179M) were cotransfected with FOXO1 or FOXO1.A3 as indicated, and analyzed for luciferase activity as described in Fig. 3. One of three independent experiments is shown (bars represent mean ± range of duplicate luciferase determinations). The average (mean ± SD; n = 3) of the ratio of fold changes for FOXO1+Akt vs FOXO1 alone was 0.5 ± 0.06. The average (mean ± SD; n = 3) of the ratio of fold changes for FOXO1.A3+Akt vs FOXO1.A3 alone was 2.1 ± 1.5.

EF1 synergizes with FOXO proteins to activate Ccng2 and Rbl2 promoters

Previous studies have shown that FOXO proteins cooperate with other transcription factors and coactivators to regulate promoter function (20, 28). Further analysis of the promoter sequences of Ccng2 and Rbl2 using the Matinspector program revealed several sites that precisely match the consensus for binding (AGGTG) by the transcription factor EF1 (other names include ZEB1, AREB6, MEB1, BZP, and zfhep) (29, 30) (Fig. 2). This zinc finger protein can be either a repressor or activator of transcription in different systems (29, 31). EF1 has been shown to function in T cell development and to repress IL-2 transcription in T cells (32, 33), but its function in B cells has not been investigated. Transfection of A20 cells with EF1 alone augmented Ccng2 and Rbl2 promoter activity 10- to 20-fold (Fig. 5A). Cotransfection of EF1 and FOXO1 or FOXO3a resulted in synergistic activation of the promoters (Fig. 5A). Supporting the idea that cooperation between FOXO proteins and EF1 is a general phenomenon in mature B cells, we observed similar effects in M12 B lymphoma cells (Fig. 5B). Importantly, the synergistic transactivation of the Ccng2 and Rbl2 promoters by FOXO1 or FOXO3a and EF1 was promoter specific, as FOXO, EF1, or the combination did not activate control constructs, including the pGL2.TKmin, pGL2.PU.1, pGL2.basic, Rbl2, or Ccng2 promoters in reverse orientation (Fig. 5C and data not shown). This demonstrates that the increased transcriptional activity from the Ccng2 and Rbl2 promoters was not due to a general increase in transcription resulting from forced expression of FOXO proteins and/or EF1. Together, these findings indicate that FOXO proteins and EF1 cooperate functionally to specifically augment transcription from the Ccng2 and Rbl2 promoters in B cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. EF1 activates the Ccng2 and Rbl2 promoters and synergizes with FOXO factors. A, A20 cells were cotransfected with the indicated reporter constructs and FOXO or/and EF1 expression vectors, and analyzed for luciferase activity as described in Fig. 3. B, Luciferase reporter assays in M12 cells, a murine IgG+ mature B cell lymphoma. C, Control promoters pGL2.TKmin and pGL2.PU.1 are not activated by FOXO and/or EF1 in A20 cells. A, The mean ± SE of three independent experiments. B and C, Data that are representative of three independent experiments with similar results (error bars represent range of duplicate measurements).

FOXO and EF1 factors bind to Ccng2 and Rbl2 promoters

We performed EMSA experiments to determine whether the consensus-binding elements for FOXO and EF1 could bind these factors in vitro. Extracts of Cos7 cells transfected with FOXO constructs were used as a source of abundant FOXO protein. These extracts formed specific binding complexes with the four IRS elements of the Ccng2 promoter, and with two IRS elements in the Rbl2 promoter (Fig. 6, A and B, and data not shown), as compared with the extracts of cells transfected by empty vector constructs. The specificity of these complexes was confirmed by incubating with Ab against FOXO3a, which caused supershifts in the Ccng2 probes and inhibited the specific binding of the Rbl2 probe. The specificity of the FOXO3a-DNA complex was also shown by competition by a 100-fold molar excess of unlabeled probe, but not by oligomers that have mutated FOXO binding sites. Extracts of 293T cells transfected with EF1 also formed specific complexes with consensus-binding elements in both the Ccng2 and Rbl2 promoters (Fig. 6C). Similarly, this specific complex could be supershifted by incubating with 9E10 Ab directed against the Myc epitope tag, and inhibited by excess unlabeled probes, but not affected by mouse preimmune serum or irrelevant oligos.

View larger version (108K): [in this window] [in a new window]   FIGURE 6. FOXO and EF1 bind to isolated promoter elements in EMSA assays, and to the endogenous promoters in ChIP assays. A, FOXO3a binds to four IRS motifs in the Ccng2 promoter. Specificity controls are shown for two of these sites. Cos7 cell extracts transfected by FOXO3.A3 or empty expression vector (–) were incubated with 32P-end-labeled oligonucleotide probes. Normal rabbit IgG (lanes 3 and 9) or Abs against FOXO3a (lanes 4 and 10) were included in the binding reactions. Specificity of the FOXO3a-DNA complex was also shown by competition of a 100-fold molar excess of unlabeled probes (lanes 5 and 11), but not by DNA oligos with mutated IRS motifs (lanes 6 and 12). A representative result of three independent experiments is shown. B, FOXO3a binds to two IRS motifs in the Rbl2 promoter. Specificity controls are shown for one of these sites. C, EF1 binds to the consensus EF1 motifs from Ccng2 and Rbl2 promoter region. Specificity controls are shown for both of these sites. 293T lysates transfected with EF1 or empty vector were used in the reactions. 9E10 Ab directed against the EF1-Myc tag supershifts the specific EF1 complex (lanes 3 and 9). D, FOXO proteins and EF1 are able to bind the Ccng2 and Rbl2 promoters in vivo. Chromatin proteins were cross-linked to DNA by formaldehyde in primary mouse B cells and immunoprecipitated by specific Abs (to FOXO1, FOXO3a, and EF1), nonimmune IgG control or no Ab (H2O). PCR in the exponential phase were detected by Southern blotting for Ccng2 and Rbl2 promoter amplicons, and by ethidium bromide staining for the CAI promoter amplicon.

FOXO-dependent promoter induction and cooperation with EF1 do not require binding to IRS elements

To determine whether these FOXO binding sites are essential for activation of the Ccng2 promoter, we made a series of truncation mutants and also used site-directed mutagenesis to specifically mutate the consensus FOXO binding sites. Mutation of individual IRS elements did not diminish promoter activation by FOXO cotransfection (data not shown). FOXO1 transfection also augmented promoter activity of a 770-bp fragment (Del5) in which only one IRS element is preserved (Fig. 7A). Furthermore, mutation of this single IRS sequence within Del5 did not abolish FOXO1-dependent activation. For each mutant construct, synergy with EF1 was at least partially maintained. Similar results were obtained using FOXO3a ± EF1 (data not shown). This raised the possibility that binding to IRS elements was not essential to the function of FOXO transcription factors in this system.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Promoter activation by FOXO proteins does not require binding to consensus IRS elements. A, Ccng2 promoter without consensus IRS sites can still be activated by FOXO1 and EF1. Reporter assays in A20 cells were done using the full-length promoter, a deletion retaining only the FOXO-c site (Del5), or the Del5 mutation in which the FOXO-c site has been mutated (Del5F) as shown above the graph. F = FOXO binding site; E = EF1 binding site. The full-length and mutant promoters had comparable basal luciferase values. B, EMSA assays were conducted using Cos7 extracts transfected by pcDNA3 empty vector (EV), FOXO1 wild type, the FOXO1(H215R) mutant that cannot bind IRS elements, and FOXO1.A3. C, FOXO1(H215R) mutant can activate the Ccng2 and Rbl2 promoter in a similar pattern as wild-type FOXO1. A20 cells were cotransfected by Ccng2 or Rbl2 full-length promoter reporter with wild-type FOXO1 or FOXO1(H215R) and/or EF1 as indicated.

EF1 augments expression of endogenous Ccng2 and Rbl2 and enhances FOXO-dependent cell cycle arrest

To assess the effect of FOXO and EF1 on endogenous gene expression, we isolated total RNA from A20 cells transfected with these factors. We used a regulatable FOXO variant (FOXO3a.A3-ER) that is expressed in an inactive form until the addition of the estrogen analog 4-hydroxytamoxifen (4-OHT) (7). RNA was prepared 64 h after transfection, with some samples receiving 4-OHT during the last 16 h. Northern blot analysis indicated that either FOXO3a.A3-ER or EF1 alone augmented expression of endogenous cyclin G₂ mRNA by 2- to 3-fold (Fig. 8, A and B). p130/Rb2 mRNA could not be detected by Northern blot under any condition (data not shown), so we used a more sensitive real-time quantitative PCR assay. The results showed that FOXO3a.A3-ER or EF1 alone enhanced levels of endogenous p130/Rb2 mRNA, similar to the effect on endogenous cyclin G₂ (Fig. 8, C and D). Induction of endogenous cyclin G₂ has been observed in FOXO-transfected fibroblasts (21), and endogenous p130/Rb2 mRNA is induced by FOXO expression in a colon carcinoma cell line (11), but the effects of EF1 on these genes have not been reported. Cotransfection of A20 cells with FOXO3a.A3-ER and EF1 resulted in a greater induction of cyclin G₂ and p130/Rb2 mRNA than that observed with either factor alone (Fig. 8, A–D).

View larger version (27K): [in this window] [in a new window]   FIGURE 8. A–D, FOXO3a and EF1 up-regulate endogenous cyclin G2 and p130 mRNA in B cells. A20 cells were transfected with pMIT-FOXO3a.A3-ER and either empty vector pMIT (lanes 1 and 2) or pMIT-EF1 (lanes 3 and 4) as indicated. Forty-eight hours later, Thy1.1-positive cells were purified by MACS column and cultured for an additional 16 h in the absence or presence of 4-OHT as indicated. Total RNA was extracted for Northern blot analysis with radiolabeled cyclin G2 and GADPH cDNA probes. Filters were exposed to phosphor screen (A) and results were quantified (B) using ImageQuant software (Amersham). Quantitation of p130 (C) and cyclin G2 (D) real-time PCR data from analysis of RNA extracted from A20 cells transfected as above. Expression relative to -actin is shown. Numbers on y-axes represent fold increase. The results are representative of two experiments. E, EF1 augments FOXO-mediated cell cycle arrest in A20 cells. Constructs for FOXO1.A3 and EF1 were transfected into A20 cells by electroporation, together or with empty vector (EV) controls. Forty-eight hours after transfection, cell cycle analysis was done by FACS, gating on live cells expressing the IRES-linked Thy1.1 marker gene. The results are representative of four experiments using FOXO1.A3 and three using FOXO3a.A3. Compared with cells transfected with empty vectors, the average (mean ± SD; n = 4) percentage of increases in G0-G1 phase of cells expressing FOXO1.A3 and FOXO1.A3+EF1 were 15 ± 3.5 and 20.5 ± 1.9, respectively (p = 0.022 for FOXO1.A3+EF1 vs FOXO1.A3 alone). The average (mean ± SD; n = 3) percentage of increases in G0-G1 phase of cells expressing FOXO3a.A3 and FOXO3a.A3+EF1 were 7 ± 1 and 12.3 ± 1.5, respectively (p = 0.057 for FOXO3a.A3+EF1 vs FOXO3a.A3 alone).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The central role of FOXO transcription factors in control of cellular proliferation, survival, stress resistance, and aging has prompted extensive investigation of FOXO target genes and mechanisms of regulation. Previous reports established the human Rbl2 gene and the murine Ccng2 gene as FOXO targets involved in quiescence of nonlymphoid cells (11, 21). In our previous work, we identified the murine Rbl2 and Ccng2 genes as candidate FOXO target genes important for quiescence in B lymphocytes. Specifically, we found that mRNA levels for these genes are high in resting B cells, and rapidly down-regulated by mitogen stimulation in a PI3K-dependent manner (15). In this study, we have provided further evidence that Rbl2 and Ccng2 are FOXO target genes in murine B cells, and have described a novel functional cooperation between FOXO proteins and the EF1 transcription factor in this context. EF1 (ZEB1) and a related protein, ZEB2, are transcriptional regulators that can either activate or repress transcription, depending on the promoter and associations with coactivators and corepressors (29, 31, 33). Gene disruption in mice identified a role for EF1 in skeletal patterning and T cell development (30, 32), but a function for EF1 in B lymphocytes has not been reported. Microarray experiments indicate that EF1 is expressed in resting splenic B cells and down-regulated following mitogenic stimulation, similar to the pattern for FOXO1 (34).

Our data show that FOXO proteins and EF1 can bind to isolated promoter elements in vitro and are bound to the Ccng2 and Rbl2 promoters in resting splenic B cells. Transfection of either FOXO or EF1 augments transcription of a reporter gene linked to either the Ccng2 or Rbl2 promoter, and cotransfection results in marked synergy that is specific for these promoters. A simple model from these data is that direct binding of both FOXO and EF1 transcription factors to the Ccng2 and Rbl2 promoters leads to synergistic effects on promoter activation. However, our data strongly suggest that direct binding of FOXO proteins to IRS elements is not essential for these effects. Mutation/deletion of the consensus IRS sequences in the Ccng2 promoter, or expression of the FOXO1(H215R) mutant, did not abrogate FOXO-induced promoter activity nor the synergy with EF1. The H215R mutation affects a critical residue in helix 3 of the DNA binding domain that contacts consensus IRS elements (35), but should not abrogate association with other transcription factors and might preserve interactions of the FOXO factor with noncanonical DNA elements. These mechanisms appear to be quite important for FOXO function, as illustrated by a global analysis of FOXO target genes in renal carcinoma cells (19). In that study, the H215R mutant had a similar influence as wild-type FOXO1 on a considerable fraction of FOXO-regulated genes. Consistent with our findings, Ccng2 (cyclin G₂) was one of the genes judged to be largely independent of IRS motif binding.

FOXO proteins are known to form complexes with other transcriptional regulatory proteins, including Smad transcription factors and the coactivator p300 (20, 28). However, in coimmunoprecipitation experiments, we did not observe physical association of FOXO proteins with EF1, even when Abs to epitope-tagged, overexpressed proteins were used (data not shown). Like FOXO proteins (28), EF1 has been reported to interact with the coactivator p300 (31). This raises the possibility that a multiprotein complex forms among FOXO, EF1, and p300. However, cotransfection of p300 did not facilitate the association of FOXO proteins with EF1 (data not shown). It is possible that physical association of FOXO and EF1 transcription factors occurs in vivo, perhaps with p300 or other cofactors, but cannot be recovered in cell extracts under the conditions tested.

The interaction of FOXO and Smad factors has been studied extensively in the context of the Cdkn1A (p21^CIP) promoter in epithelial cells (20). These factors bind to adjacent elements in the promoter and the integrity of both the FOXO binding element and the Smad binding site is required for TGF to activate Cdkn1A transcription. In addition, a FOXO4 DNA binding mutant equivalent to FOXO1(H215R) does not activate the Cdkn1A promoter. These findings contrast with our analysis of FOXO/EF1 cooperation at B cell target promoters. One possible explanation for the equivalent function of wild-type and FOXO1(H215R) in our system is that FOXO function could be mediated mainly through EF1 and its DNA binding sites. However, mutation of the canonical EF1 motif 3' to the FOXO-c element within the Del5 fragment of the Ccng2 promoter (Fig. 1B) did not abrogate Del5 activation either by FOXO, EF1 or the combination, even when the IRS sequence was also mutated (data not shown). It remains possible that nonconsensus binding sites for FOXO and/or EF1 are sufficient to mediate cooperative promoter activation. The ChIP experiments indicate that FOXO proteins and EF1 are associated with the endogenous Ccng2 and Rbl2 promoters in vivo, but do not establish whether the binding is direct. Additional experiments are necessary to define the cis-acting elements essential for activation of these promoters by FOXO proteins and EF1.

Supporting a role for EF1 in regulation of the endogenous Ccng2 and Rbl2 promoters, transfection of A20 cells with EF1 increased the mRNA levels for these two genes. EF1 was not able to induce cell cycle arrest/delay on its own in A20 cells, but did enhance the effect of FOXO1. It is possible that FOXO factors regulate additional genes independently of EF1 that are required to suppress cell cycle progression in A20 cells. We were not able to generate high titer retroviruses expressing EF1, despite many attempts and numerous vectors. This might be due to effects of EF1 on the viability or retroviral packaging ability of 293T cells. Consequently, we could not study functional cooperation in primary B cells, which are refractory to other methods of gene delivery.

Expression of FOXO3a.A3-ER, followed by 4-OHT induction, resulted in augmented mRNA levels of Ccng2 and Rbl2 compared with cells cultured in the absence of the inducer. Expression of EF1 with induced FOXO3a.A3-ER produced an additional increase in expression of the endogenous gene(s). These data support the conclusion that Ccng2 and Rbl2 are bona fide FOXO target gene(s) in B cells and that EF1 can cooperate with FOXO factors to augment expression. The effect of FOXO/EF1 coexpression on endogenous target genes was less dramatic than observed using promoter-reporter assays. This could be explained by the fact that Northern blot analysis and RT-PCR measure steady-state levels of mRNA at a single time point, whereas the reporter assay measures accumulation of the protein product driven by the promoter over the time since transfection occurred.

In summary, we have established a novel functional cooperation between FOXO transcription factors and EF1. These proteins synergize to activate promoters of two genes with known antiproliferative properties, Ccng2 and Rbl2. These findings suggest that the programming of lymphocyte quiescence by FOXO factors might require coordinated interactions with EF1 and other transcription factors at target promoters.

	Acknowledgments

 
We thank Marian Waterman and Chris Hughes for comments on the manuscript, Tim Osborne for the p300 cDNA and advice on transcription experiments, Xiaocui Zhu for help with microarray data analysis, and Travis Moore for help with mouse colony maintenance. We also thank the following investigators for providing DNA constructs: Boudewijn Burgering, Anne Brunet and Kun-Liang Guan (FOXO), Elizabeth Conner (cyclin G₂), Antonio Giordano (p130), Yujiro Higashi (EF1), and Timothy Bender (carbonic anhydrase ChIP primers).

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work is supported by National Institutes of Health Grant AI50831 (to D.A.F.) and American Cancer Society Research Scholar Grant 05-143-01-LIB (to D.A.F.).

² Address correspondence and reprint requests to Dr. David A. Fruman, Center for Immunology, and Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900. E-mail address: dfruman{at}uci.edu

³ Abbreviations used in this paper: IRS, insulin response sequence; ChIP, chromatin immunoprecipitation.

Received for publication September 2, 2005. Accepted for publication November 25, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Accili, D., K. C. Arden. 2004. FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117: 421-426. [Medline]
2. Burgering, B. M., G. J. Kops. 2002. Cell cycle and death control: long live forkheads. Trends Biochem. Sci. 27: 352-360. [Medline]
3. Tran, H., A. Brunet, E. C. Griffith, M. E. Greenberg. 2003. The many forks in FOXO’s road. Sci. STKE 2003: RE5[Medline]
4. Furuyama, T., K. Kitayama, Y. Shimoda, M. Ogawa, K. Sone, K. Yoshida-Araki, H. Hisatsune, S.-i. Nishikawa, K. Nakayama, K. Nakayama, et al 2004. Abnormal angiogenesis in foxo1 (Fkhr)-deficient mice. J. Biol. Chem. 279: 34741-34749. [Abstract/Free Full Text]
5. Hosaka, T., W. H. Biggs, III, D. Tieu, A. D. Boyer, N. M. Varki, W. K. Cavenee, K. C. Arden. 2004. Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc. Natl. Acad. Sci. USA 101: 2975-2980. [Abstract/Free Full Text]
6. Lin, L., J. D. Hron, S. L. Peng. 2004. Regulation of NF-B, Th activation, and autoinflammation by the forkhead transcription factor Foxo3a. Immunity 21: 203-213. [Medline]
7. Dijkers, P. F., R. H. Medema, C. Pals, L. Banerji, N. S. Thomas, E. W. Lam, B. M. Burgering, J. A. Raaijmakers, J. W. Lammers, L. Koenderman, P. J. Coffer. 2000. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27^KIP1. Mol. Cell. Biol. 20: 9138-9148. [Abstract/Free Full Text]
8. Kharas, M. G., J. A. Deane, S. Wong, K. R. O’Bosky, N. Rosenberg, O. N. Witte, D. A. Fruman. 2004. Phosphoinositide 3-kinase signaling is essential for ABL oncogene-mediated transformation of B-lineage cells. Blood 103: 4268-4275. [Abstract/Free Full Text]
9. Stahl, M., P. F. Dijkers, G. J. Kops, S. M. Lens, P. J. Coffer, B. M. Burgering, R. H. Medema. 2002. The forkhead transcription factor FoxO regulates transcription of p27^Kip1 and Bim in response to IL-2. J. Immunol. 168: 5024-5031. [Abstract/Free Full Text]
10. Kops, G. J., T. B. Dansen, P. E. Polderman, I. Saarloos, K. W. Wirtz, P. J. Coffer, T. T. Huang, J. L. Bos, R. H. Medema, B. M. Burgering. 2002. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419: 316-321. [Medline]
11. Kops, G. J., R. H. Medema, J. Glassford, M. A. Essers, P. F. Dijkers, P. J. Coffer, E. W. Lam, B. M. Burgering. 2002. Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol. Cell. Biol. 22: 2025-2036. [Abstract/Free Full Text]
12. Yusuf, I., D. A. Fruman. 2003. Regulation of quiescence in lymphocytes. Trends Immunol. 24: 380-386. [Medline]
13. Deane, J. A., D. A. Fruman. 2004. Phosphoinositide 3-kinase: diverse roles in immune cell activation. Annu. Rev. Immunol. 22: 563-598. [Medline]
14. Okkenhaug, K., B. Vanhaesebroeck. 2003. PI3K in lymphocyte development, differentiation and activation. Nat. Rev. Immunol. 3: 317-330. [Medline]
15. Yusuf, I., X. Zhu, M. G. Kharas, J. Chen, D. A. Fruman. 2004. Optimal B-cell proliferation requires phosphoinositide 3-kinase-dependent inactivation of FOXO transcription factors. Blood 104: 784-787. [Abstract/Free Full Text]
16. Fruman, D. A., G. Z. Ferl, S. S. An, A. C. Donahue, A. B. Satterthwaite, O. N. Witte. 2002. Phosphoinositide 3-kinase and Bruton’s tyrosine kinase regulate overlapping sets of genes in B lymphocytes. Proc. Natl. Acad. Sci. USA 99: 359-364. [Abstract/Free Full Text]
17. Furuyama, T., T. Nakazawa, I. Nakano, N. Mori. 2000. Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues. Biochem. J. 349: 629-634. [Medline]
18. Lee, S. S., S. Kennedy, A. C. Tolonen, G. Ruvkun. 2003. DAF-16 target genes that control C. elegans life-span and metabolism. Science 300: 644-647. [Abstract/Free Full Text]
19. Ramaswamy, S., N. Nakamura, I. Sansal, L. Bergeron, W. R. Sellers. 2002. A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2: 81-91. [Medline]
20. Seoane, J., H.-V. Le, L. Shen, S. A. Anderson, J. Massague. 2004. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117: 211-223. [Medline]
21. Martinez-Gac, L., M. Marques, Z. Garcia, M. R. Campanero, A. C. Carrera. 2004. Control of cyclin G₂ mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead. Mol. Cell. Biol. 24: 2181-2189. [Abstract/Free Full Text]
22. Miglarese, M. R., A. F. Richardson, N. Aziz, T. P. Bender. 1996. Differential regulation of c-Myb-induced transcription activation by a phosphorylation site in the negative regulatory domain. J. Biol. Chem. 271: 22697-22705. [Abstract/Free Full Text]
23. Chen, J., C. S. Kremer, T. P. Bender. 2002. A Myb dependent pathway maintains Friend murine erythroleukemia cells in an immature and proliferating state. Oncogene 21: 1859-1869. [Medline]
24. Bennin, D. A., A. S. Don, T. Brake, J. L. McKenzie, H. Rosenbaum, L. Ortiz, A. A. DePaoli-Roach, M. C. Horne. 2002. Cyclin G₂ associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G₁-S phase cell cycle arrest. J. Biol. Chem. 277: 27449-27467. [Abstract/Free Full Text]
25. Hansen, K., T. Farkas, J. Lukas, K. Holm, L. Ronnstrand, J. Bartek. 2001. Phosphorylation-dependent and -independent functions of p130 cooperate to evoke a sustained G₁ block. EMBO J. 20: 422-432. [Medline]
26. Deleted in proof.
27. Van Der Heide, L. P., M. F. Hoekman, M. P. Smidt. 2004. The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem. J. 380: 297-309. [Medline]
28. Nasrin, N., S. Ogg, C. M. Cahill, W. Biggs, S. Nui, J. Dore, D. Calvo, Y. Shi, G. Ruvkun, M. C. Alexander-Bridges. 2000. DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells. Proc. Natl. Acad. Sci. USA 97: 10412-10417. [Abstract/Free Full Text]
29. Fontemaggi, G., A. Gurtner, S. Strano, Y. Higashi, A. Sacchi, G. Piaggio, G. Blandino. 2001. The transcriptional repressor ZEB regulates p73 expression at the crossroad between proliferation and differentiation. Mol. Cell. Biol. 21: 8461-8470. [Abstract/Free Full Text]
30. Takagi, T., H. Moribe, H. Kondoh, Y. Higashi. 1998. EF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages. Development 125: 21-31. [Abstract]
31. Postigo, A. A., J. L. Depp, J. J. Taylor, K. L. Kroll. 2003. Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. EMBO J. 22: 2453-2462. [Medline]
32. Higashi, Y., H. Moribe, T. Takagi, R. Sekido, K. Kawakami, H. Kikutani, H. Kondoh. 1997. Impairment of T cell development in EF1 mutant mice. J. Exp. Med. 185: 1467-1480. [Abstract/Free Full Text]
33. Yasui, D. H., T. Genetta, T. Kadesch, T. M. Williams, S. L. Swain, L. V. Tsui, B. T. Huber. 1998. Transcriptional repression of the IL-2 gene in Th cells by ZEB. J. Immunol. 160: 4433-4440. [Abstract/Free Full Text]
34. Zhu, X., R. Hart, M. S. Chang, J. W. Kim, S. Y. Lee, Y. A. Cao, D. Mock, E. Ke, B. Saunders, A. Alexander, et al 2004. Analysis of the major patterns of B cell gene expression changes in response to short-term stimulation with 33 single ligands. J. Immunol. 173: 7141-7149. [Abstract/Free Full Text]
35. Tang, E. D., G. Nunez, F. G. Barr, K. L. Guan. 1999. Negative regulation of the forkhead transcription factor FKHR by Akt. J. Biol. Chem. 274: 16741-16746. [Abstract/Free Full Text]

This article has been cited by other articles:

				R. M. Hinman, W. A. Nichols, T. M. Diaz, T. D. Gallardo, D. H. Castrillon, and A. B. Satterthwaite Foxo3-/- mice demonstrate reduced numbers of pre-B and recirculating B cells but normal splenic B cell sub-population distribution Int. Immunol., July 1, 2009; 21(7): 831 - 842. [Abstract] [Full Text] [PDF]

				S. Fabre, F. Carrette, J. Chen, V. Lang, M. Semichon, C. Denoyelle, V. Lazar, N. Cagnard, A. Dubart-Kupperschmitt, M. Mangeney, et al. FOXO1 Regulates L-Selectin and a Network of Human T Cell Homing Molecules Downstream of Phosphatidylinositol 3-Kinase J. Immunol., September 1, 2008; 181(5): 2980 - 2989. [Abstract] [Full Text] [PDF]

				T. Hidaka, S. Nakahata, K. Hatakeyama, M. Hamasaki, K. Yamashita, T. Kohno, Y. Arai, T. Taki, K. Nishida, A. Okayama, et al. Down-regulation of TCF8 is involved in the leukemogenesis of adult T-cell leukemia/lymphoma Blood, July 15, 2008; 112(2): 383 - 393. [Abstract] [Full Text] [PDF]

				T. Pew, M. Zou, D. R. Brickley, and S. D. Conzen Glucocorticoid (GC)-Mediated Down-Regulation of Urokinase Plasminogen Activator Expression via the Serum and GC Regulated Kinase-1/Forkhead Box O3a Pathway Endocrinology, May 1, 2008; 149(5): 2637 - 2645. [Abstract] [Full Text] [PDF]

				I. Yusuf, M. G. Kharas, J. Chen, R. Q. Peralta, A. Maruniak, P. Sareen, V. W. Yang, K. H. Kaestner, and D. A. Fruman KLF4 is a FOXO target gene that suppresses B cell proliferation Int. Immunol., May 1, 2008; 20(5): 671 - 681. [Abstract] [Full Text] [PDF]

				X.-F. Le, A. S. Arachchige-Don, W. Mao, M. C. Horne, and R. C. Bast Jr. Roles of human epidermal growth factor receptor 2, c-jun NH2-terminal kinase, phosphoinositide 3-kinase, and p70 S6 kinase pathways in regulation of cyclin G2 expression in human breast cancer cells Mol. Cancer Ther., November 1, 2007; 6(11): 2843 - 2857. [Abstract] [Full Text] [PDF]

				K. L. Good and S. G. Tangye Decreased expression of Kruppel-like factors in memory B cells induces the rapid response typical of secondary antibody responses PNAS, August 14, 2007; 104(33): 13420 - 13425. [Abstract] [Full Text] [PDF]

				R. M. Hinman, J. N. Bushanam, W. A. Nichols, and A. B. Satterthwaite B Cell Receptor Signaling Down-Regulates Forkhead Box Transcription Factor Class O 1 mRNA Expression via Phosphatidylinositol 3-Kinase and Bruton's Tyrosine Kinase J. Immunol., January 15, 2007; 178(2): 740 - 747. [Abstract] [Full Text] [PDF]

				M. G. Kharas, I. Yusuf, V. M. Scarfone, V. W. Yang, J. A. Segre, C. S. Huettner, and D. A. Fruman KLF4 suppresses transformation of pre-B cells by ABL oncogenes Blood, January 15, 2007; 109(2): 747 - 755. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, J. Articles by Fruman, D. A. Search for Related Content PubMed PubMed Citation Articles by Chen, J. Articles by Fruman, D. A.