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© 2015 Macmillan Publishers Limited. All rights reserved., T-cell acute lymphoblastic leukemia (T-ALL) frequently involves aberrant expression of TAL1 (T-cell acute lymphocytic leukemia 1) and LMO2, oncogenic members of the TAL1 transcriptional complex. Transcriptional activity of the TAL1-complex is thought to have a pivotal role in the transformation of thymocytes and is associated with a differentiation block and self-renewal. The transcription factor Forkhead Box P3 (FOXP3) was recently described to be expressed in a variety of malignancies including T-ALL. Here we show that increased FOXP3 levels negatively correlate with expression of genes regulated by the oncogenic TAL1-complex in human T-ALL patient samples as well as a T-ALL cell line ectopically expressing FOXP3. In these cells, FOXP3 expression results in altered regulation of cell cycle progression and reduced cell viability. Finally, we demonstrate that FOXP3 binds LMO2 in vitro, resulting in decreased interaction between LMO2 and TAL1, providing a molecular mechanism for FOXP3-mediated transcriptional modulation in T-ALL. Collectively, our findings provide initial evidence for a novel role of FOXP3 as a tumor suppressor in T-ALL through modulation of TAL1 transcriptional activity., VF and JvL were supported by a grant from the Dutch Arthritis Foundation (Rheumafonds), and BAC was supported by a fellowship from Fundação para a Ciência e a Tecnologia. This work was supported by grant from the Dutch Arthritis Foundation (Rheumafonds), and a fellowship from Fundação para a Ciência e a Tecnologia.

SummaryStable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.

Maintenance of Foxp3 protein expression in regulatory T cells (Treg) is crucial for a balanced immune response. We have previously demonstrated that Foxp3 protein stability can be regulated through acetylation, however the specific mechanisms underlying this observation remain unclear. Here we demonstrate that SIRT1 a member of the lysine deacetylase Sirtuin (SIRT) family, but not the related SIRTs 2–7, co-localize with Foxp3 in the nucleus. Ectopic expression of SIRT1, but not SIRTs 2–7 results in decreased Foxp3 acetylation, while conversely inhibition of endogenous SIRT activity increased Foxp3 acetylation. We show that SIRT1 inhibition decreases Foxp3 poly-ubiquitination, thereby increasing Foxp3 protein levels. Co-transfection of SIRT1 with Foxp3 results in increased Foxp3 proteasomal degradation, while SIRT inhibition increases FOXP3 transcriptional activity in human Treg. Taken together, these data support a central role for SIRT1 in the regulation of Foxp3 protein levels and thereby in regulation of Treg suppressive capacity. Pharmacological modulation of SIRT1 activity in Treg may therefore provide a novel therapeutic strategy for controlling immune responses.

Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm- and mesoderm-like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c-src kinase activity is enhanced in the RPTP alpha-transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c-src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c-src in the differentiation process.

Summary Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.

Modification of proteins by ubiquitin plays a major role in a broad array of biological processes. Reversal of this process through deubiquitylation likely represents an important regulatory step in the maintenance of cellular homeostasis. However, the biological functions of deubiquitylating enzymes still remain poorly characterized. To investigate the biological role of the herpes virus-associated ubiquitin-specific protease HAUSP/USP7, we have generated stably transfected cells carrying inducible shRNA expression plasmids. USP7 mRNA and protein were strongly down-regulated 48-72 h after shRNA induction. We used a selected clone to compare whole-cell proteomes by 2D-SDS-PAGE before and after knockdown of USP7. Alterations in 36 proteins were detected and their identities were revealed by mass spectrometry analysis. Components of the replication machinery, DNA/RNA binding proteins, enzymes involved in apoptosis and metabolism were found to be down-regulated upon USP7 removal, representing proteins that are either more rapidly turned over or synthesized less efficiently in the absence of USP7-mediated deubiquitylation. Alix/HP95, a protein implicated in endosomal organization and virus budding, was confirmed by immunoblotting to become down-regulated when USP7 levels were reduced. Our results extend the current list of USP7-dependent biological processes and suggest a role for this enzyme not only in transcriptional regulation but also in DNA replication, apoptosis, and possibly endosomal organization.

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27(KIP1) through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27(KIP1) promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27(KIP1) appears to be critical in the regulation of cell survival since mere ectopic expression of p27(KIP1) was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27(KIP1) null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27(KIP1) transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

SummaryFoxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.

We have isolated the murine homologs of the members of the COUP-family of steroid hormone receptors, COUP-TF1, ARP-1 and EAR2. The proteins encoded by the murine genes appeared to be highly conserved when compared to their human counterparts. The expression of COUP-TF1 and ARP-1 was induced during differentiation of P19 embryonal carcinoma (EC) cells into derivatives of all three germ layers. Retinoic acid (RA) treatment rapidly induced expression of both genes, while other methods of differentiation were less effective. Undifferentiated P19 cells were found to express EAR2 mRNA and the expression level was only slightly elevated by RA-treatment. In addition, we analyzed the expression in P19 cells of three members of the retinoid X receptor (RXR) family, which have been shown to heterodimerize with members of the COUP-family. During RA mediated differentiation of P19 cells, RXR alpha expression was induced while RXR beta expression was not modulated and RXR gamma expression was down regulated. Gel shift analysis revealed that in P19 cells the members of the COUP-family comprise the major portion of proteins binding to a RA-responsive direct repeat of the consensus steroid hormone receptor binding half site (AGGTCA) spaced by one nucleotide (DR + 1). The members of the COUP-family appeared to down regulate RA-induced activation of RA-response element-containing reporter constructs in a promoter context-dependent manner. The expression patterns of COUP-TF1, ARP-1 and EAR2 during development were investigated by in situ hybridization. In agreement with the results obtained in vitro, the three genes appeared to be expressed in tissues derived from all three germ layers. However, COUP-TF1 and ARP-1 were found to be expressed predominantly in the developing central nervous system in mutually exclusive domains. Furthermore, strong ARP-1 expression was detected in lung and kidney. Our data strongly suggest an important role for the members of the COUP-family in the hormonal control of gene expression regulating embryogenesis.

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.