A new Drosophila APC homologue associated with adhesive zones of epithelial cells.

Adenomatous polyposis coli protein (APC) is an important tumour suppressor in the human colon epithelium. In a complex with glycogen synthase kinase-3 (GSK-3), APC binds to and destabilizes cytoplasmic ('free') beta-catenin. Here, using a yeast two-hybrid screen for proteins that bind to the Drosophila beta-catenin homologue, Armadillo, we identify a new Drosophila APC homologue, E-APC. E-APC also binds to Shaggy, the Drosophila GSK-3 homologue. Interference with E-APC function produces embryonic phenotypes like those of shaggy mutants. Interestingly, E-APC is concentrated in apicolateral adhesive zones of epithelial cells, along with Armadillo and E-cadherin, which are both integral components of the adherens junctions in these zones. Various mutant conditions that cause dissociation of E-APC from these zones also obliterate the segmental modulation of free Armadillo levels that is normally induced by Wingless signalling. We propose that the Armadillo-destabilizing protein complex, consisting of E-APC, Shaggy, and a third protein, Axin, is anchored in adhesive zones, and that Wingless signalling may inhibit the activity of this complex by causing dissociation of E-APC from these zones.

Pontin is a critical regulator for AML1-ETO-induced leukemia.

The oncogenic fusion protein AML1-ETO, also known as RUNX1-RUNX1T1 is generated by the t(8;21)(q22;q22) translocation, one of the most frequent chromosomal rearrangements in acute myeloid leukemia (AML). Identifying the genes that cooperate with or are required for the oncogenic activity of this chimeric transcription factor remains a major challenge. Our previous studies showed that Drosophila provides a genuine model to study how AML1-ETO promotes leukemia. Here, using an in vivo RNA interference screen for suppressors of AML1-ETO activity, we identified pontin/RUVBL1 as a gene required for AML1-ETO-induced lethality and blood cell proliferation in Drosophila. We further show that PONTIN inhibition strongly impaired the growth of human t(8;21)(+) or AML1-ETO-expressing leukemic blood cells. Interestingly, AML1-ETO promoted the transcription of PONTIN. Moreover, transcriptome analysis in Kasumi-1 cells revealed a strong correlation between PONTIN and AML1-ETO gene signatures and demonstrated that PONTIN chiefly regulated the expression of genes implicated in cell cycle progression. Concordantly, PONTIN depletion inhibited leukemic self-renewal and caused cell cycle arrest. All together our data suggest that the upregulation of PONTIN by AML1-ETO participate in the oncogenic growth of t(8;21) cells.

Drosophila CBP represses the transcription factor TCF to antagonize Wingless signalling.

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signalling. When signalling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Here we report that Drosophila CREB-binding protein (dCBP) binds to dTCF. dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, our data show that dCBP acetylates a conserved lysine in the Armadillo-binding domain of dTCF, and that this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, our data show that CBP represses TCF.

A function of CBP as a transcriptional co-activator during Dpp signalling.

CBP/p300 is a transcriptional co-activator that is recruited to enhancers by various DNA-binding proteins, including proteins whose activity is controlled by extracellular signals. Here, we report that Drosophila CBP loss-of-function mutants show specific defects which mimic those seen in mutants that lack the extracellular signal Dpp or its effector Mad. Furthermore, we find that CBP loss severely compromises the ability of Dpp target enhancers to respond to endogenous or exogenous Dpp. Finally, we show that CBP binds to the C-terminal domain of Mad. Our results provide evidence that CBP functions as a co-activator during Dpp signalling, and they suggest that Mad may recruit CBP to effect the transcriptional activation of Dpp-responsive genes during development.

The control of beta-catenin and TCF during embryonic development and cancer.

The Wnt signaling pathway functions reiteratively during animal development to control cell fate decisions. Inappropriate deregulation of this pathway leads to cancer in a number of tissues. The components that transduce the Wnt signal from the cell membrane to the cell nucleus are well conserved between vertebrates and Drosophila. A pivotal Wnt effector is the protein beta-catenin/Armadillo whose stability in the cytoplasm is low in unstimulated cells. Beta-catenin/Armadillo is targetted for proteasome-mediated degradation by a protein complex to which it binds. This complex consists of Axin, a putative scaffold protein which also binds to the tumor suppressor Adenomatous polyposis coli (APC) and glycogen synthase kinase 3 (GSK3)/Shaggy. Wnt signaling somehow inhibits the kinase activity of the quaternary complex. As a consequence, beta-catenin/Armadillo accumulates in the cytoplasm, translocates to the nucleus and becomes a transcriptional co-activator of T cell factor (TCF), the ultimate nuclear target of Wnt signaling. TCF is an architectural protein, mediating the assembly of multi-protein enhancer complexes. It cooperates with other enhancer-binding proteins and, together with beta-catenin/Armadillo, stimulates the transcription of Wnt target genes. Recently, repressors have been identified that prevent TCF from being active in the absence of Wnt signaling.

Teashirt is required for transcriptional repression mediated by high Wingless levels.

During development, extracellular signals often act at multiple thresholds to specify distinct transcriptional and cellular responses. For example, in the embryonic midgut of Drosophila, low Wingless levels stimulate the transcription of homeotic genes whereas high Wingless levels repress these genes. Wingless- mediated transcriptional activation is conferred by Drosophila: T-cell factor (dTCF) and its co-activator Armadillo, but the nuclear factors mediating transcriptional repression are unknown. Here we show that teashirt is required for Wingless-mediated repression of Ultrabithorax: in the midgut. Teashirt is also a repressor of the homeotic gene labial in this tissue. Furthermore, the target sequence for Tsh within the Ultrabithorax: midgut enhancer coincides with the response sequence for Wingless-mediated repression. Finally, we demonstrate that the zinc finger protein Teashirt behaves as a transcriptional repressor in transfected mammalian cells. It thus appears that the response to high Wingless levels in the Drosophila: midgut is indirect and based on transcriptional activation of the Teashirt repressor.

Epstein-Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA.

Following infection by Epstein-Barr virus (EBV), the production of viral nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C and the viral membrane protein LMP1 is essential for the permanent proliferation of primary B lymphocytes to occur. Among these, the transcription factor EBNA2 is central to the immortalizing process, since it activates not only the transcription of all the EBNA proteins and LMP1, TP1, and TP2 but also certain cellular genes. EBNA2 is targeted to its DNA-responsive elements through direct interaction with the DNA-binding cellular repressor RBP-J kappa. In a transient-expression assay, the EBNA2-activated transcription was found to be downregulated by EBNA3A, EBNA3B, and EBNA3C. However, since it has been reported that EBNA3C, but not EBNA3A, directly contacts RBP-J kappa in vitro, these proteins appear to repress through different mechanisms. Here, we report for the first time that EBNA3A and EBNA3C both stably interact with RBP-J kappa and most probably repress EBNA2-activated transcription by destabilizing the binding of RBP-J kappa to DNA.

RBP-J kappa repression activity is mediated by a co-repressor and antagonized by the Epstein-Barr virus transcription factor EBNA2.

The Epstein-Barr virus (EBV) protein EBNA2 is a transcriptional activator that can be targeted to its DNA responsive elements by direct interaction with the cellular protein RBP-J kappa. RBP-J kappa is a ubiquitous factor, highly conserved between man, mouse and Drosophila, whose function in mammalian cells is largely unknown. Here we provide evidence that RBP-J kappa is a transcriptional repressor and, more importantly, that RBP-J kappa repression is mediated by a co-repressor. The function of the co-repressor could be counterbalanced by making a fusion protein (RBP-VP16) between RBP-J kappa and the VP16 activation domain. This RBP-VP16-mediated activation could be strongly increased by an EBNA2 protein deprived of its activation domain, but not by an EBNA2 protein incapable of making physical contact with RBP-J kappa. Our results suggest that EBNA2 activates transcription by both interfering with the function of a co-repressor recruited by RBP-J kappa and providing an activation domain.

Transcriptional repression by the Epstein-Barr virus EBNA3A protein tethered to DNA does not require RBP-Jkappa.

The Epstein-Barr virus (EBV) proteins EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1 and EBNA-LP are essential for the in vitro immortalization of primary B lymphocytes by EBV. EBNA2 is a transcriptional activator of viral and cellular genes. Both EBNA3A and EBNA3C have been shown to specifically inhibit EBNA2-activated transcription by direct interaction with RBP-Jkappa, a cellular DNA-binding factor known to recruit EBNA2 to EBNA2-responsive genes. This interaction interferes with the binding of RBP-Jkappa to DNA in vitro, and this is probably the mechanism by which EBNA3A and EBNA3C repress EBNA2-activated transcription in vivo. EBNA3A and EBNA3C also directly repress transcription when tethered to a promoter via the DNA-binding domain of the yeast Gal4 protein. As RBP-Jkappa has been previously shown to be a repressor in mammalian cells, this repression could be due to the recruitment of RBP-Jkappa by Gal4-EBNA3A and 3C. In this study, we have precisely mapped the domain of EBNA3A involved in the interaction with RBP-Jkappa and we have shown that interaction with RBP-Jkappa is not required for the Gal4-EBNA3A-mediated repression. Furthermore, we have characterized in EBNA3A a domain of 143 amino acids which is necessary and sufficient for EBNA3A-dependent repression.

The human J kappa recombination signal sequence binding protein (RBP-J kappa) targets the Epstein-Barr virus EBNA2 protein to its DNA responsive elements.

The Epstein-Barr virus (EBV) protein EBNA2, which is essential for the immortalization of human primary B cells by EBV, acts as a transcriptional activator of cellular and viral genes. Specific responsive elements have been characterized in several of the promoters activated by EBNA2. They all share the core sequence GTGGGAA. EBNA2 does not, however, bind to these sequences directly, but appears to be targeted to them by a cellular protein. A similar core sequence has recently been identified as a high-affinity binding site for the human recombination signal sequence binding protein RBP-J kappa. Here we provide evidence that RBP-J kappa binds to specific sequences in EBNA2-responsive elements. Our results also demonstrate that RBP-J kappa makes direct physical contact with EBNA2 in solution and recruits EBNA2 to its cognate DNA sequences, suggesting that RBP-J kappa may mediate EBNA2 transactivation of both cellular and viral genes.