The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression.

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.

Systematic interactome mapping of acute lymphoblastic leukemia cancer gene products reveals EXT-1 tumor suppressor as a Notch1 and FBWX7 common interactor.

BACKGROUND: Perturbed genotypes in cancer can now be identified by whole genome sequencing of large number of diverse tumor samples, and observed gene mutations can be used for prognosis and classification of cancer subtypes. Although mutations in a few causative genes are directly linked to key signaling pathways perturbation, a global understanding of how known cancer genes drive oncogenesis in human is difficult to assess. METHODS: We collected available information about mutated genes in Acute Lymphoblastic Leukemia (ALL). Validated human protein interactions (PPI) were collected from IntAct, HPRD and BioGRID interactomics databases, or obtained using yeast two-hybrid screening assay. RESULTS: We have mapped interconnections between 116 cancer census gene products associated with ALL. Combining protein-protein interactions data and cancer-specific gene mutations information, we observed that 63 ALL-gene products are interconnected and identified 37 human proteins interacting with at least 2 ALL-gene products. We highlighted exclusive and coexistence genetic alterations in key signaling pathways including the PI3K/AKT and the NOTCH pathways. We then used different cell lines and reporter assay systems to validate the involvement of EXT1 in the Notch pathway. CONCLUSION: We propose that novel ALL-gene candidates can be identified based on their functional association with well-known cancer genes. We identified EXT1, a gene not previously linked to ALL via mutations, as a common interactor of NOTCH1 and FBXW7 regulating the NOTCH pathway in an FBXW7-dependend manner.

Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression. RESULTS: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway. CONCLUSIONS: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.

Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program.

The Tax oncoprotein encoded by the human T-cell leukemia virus type 1 plays a pivotal role in viral persistence and pathogenesis. Human T-cell leukemia virus type 1-infected cells proliferate faster than normal lymphocytes, expand through mitotic division, and accumulate genomic lesions. Here, we show that Tax associates with the minichromosome maintenance MCM2-7 helicase complex and localizes to origins of replication. Tax modulates the spatiotemporal program of origin activation and fires supplementary origins at the onset of S phase. Thereby, Tax increases the DNA replication rate, accelerates S phase progression, but also generates a replicative stress characterized by the presence of genomic lesions. Mechanistically, Tax favors p300 recruitment and histone hyperacetylation at late replication domains, advancing their replication timing in early S phase.

DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Intact intracellular tail is critical for proper functioning of the tumor-associated, hypoxia-regulated carbonic anhydrase IX.

Carbonic anhydrase IX (CA IX) is a tumor-associated, hypoxia-induced enzyme involved in pH regulation and cell adhesion. Its catalytically active ectodomain (ECD) is linked to a transmembrane region and a short intracellular (IC) tail. Removal of the IC tail causes intracellular localization of CA IX. Mutations of basic amino acids within IC do not perturb the membrane position, but reduce shedding of the CA IX ectodomain as well as CA IX-mediated cell dissociation. Moreover, they abolish the CA IX capacity to acidify extracellular pH (pHe) and bind CA IX-selective sulfonamide inhibitor in hypoxia. These findings provide the first evidence for the critical contribution of the IC tail to the proper functioning of CA IX.

Earlier onset of delta-retrovirus-induced leukemia after splenectomy.

Infection by delta-retroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) is mostly asymptomatic. Indeed, only a minority (<5%) of delta-retrovirus infected hosts will develop either lymphoproliferative or neurodegenerative diseases after long latency periods. In fact, the host immune response is believed to tightly control viral replication but this assumption has not been definitely proven in vivo. Here, we provide direct experimental evidence demonstrating that integrity of the spleen is required to control pathogenesis. In the BLV model, we show that asplenia decreases efficiency of the immune response and induces an imbalance in cell dynamics resulting in accelerated onset of leukemia. These observations enlighten a potential threat in splenectomized HTLV-1 carriers and justify a regular preventive evaluation.

Protein-protein interactions and gene expression regulation in HTLV-1 infected cells.

Human T-cell leukemia virus type 1 (HTLV-1) propagates in CD4 or CD8 T cells and, after extended latency periods of 30-50 years, causes a rapidly fatal leukemia called adult T-cell leukemia/lymphoma (ATL). Infection with HTLV-1 is also associated with a degenerative neuromuscular disease referred to as tropical spastic paraparesis or HTLV-1-associated myelopathy. HTLV genome, in addition to the structural proteins and retroviral enzymes, codes for a region at its 3' end originally designated pX. The products of this region (Tax, Rex, p12I, p13II, p30II and HBZ) play important roles in deregulation of cellular functions by either directly disrupting cellular factors or altering transcription of viral and cellular genes. Here, we will review current knowledge of protein-protein interactions that regulate transcriptional functions of proteins encoded by the pX region.

The HTLV-1 Tax interactome.

The Tax1 oncoprotein encoded by Human T-lymphotropic virus type I is a major determinant of viral persistence and pathogenesis. Tax1 affects a wide variety of cellular signalling pathways leading to transcriptional activation, proliferation and ultimately transformation. To carry out these functions, Tax1 interacts with and modulates activity of a number of cellular proteins. In this review, we summarize the present knowledge of the Tax1 interactome and propose a rationale for the broad range of cellular proteins identified so far.

Emphasis on cell turnover in two hosts infected by bovine leukemia virus: a rationale for host susceptibility to disease.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects and induces accumulation of B-lymphocytes in the peripheral blood and lymphoid tissues of cattle, leading to leukemia/lymphoma. BLV can also be experimentally transmitted to sheep, in which disease appears earlier and at higher frequencies. Abnormal accumulation of leukemic B-lymphocytes results from an alteration of different parameters that include cell proliferation and death as well as migration to lymphoid tissues. Interestingly, B lymphocyte turnover is increased in BLV-infected sheep but reduced in cattle, revealing a potential relationship between cell kinetics and disease progression.

Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep.

Based on a reverse genetics approach, we previously reported that bovine leukemia virus (BLV) mutants harboring deletions in the accessory R3 and G4 genes persist at very low proviral loads and are unable to induce leukemia or lymphoma in sheep, indicating that these R3 and G4 gene sequences are required for pathogenesis. We now show that lymphoma can occur, albeit infrequently (1 case of 20) and after extended periods of latency (7 years). Direct sequencing and reinfection experiments demonstrated that lymphomagenesis was not due to the reversion of the mutant to the wild type. Similar observations with another type of attenuated mutant impaired in the transmembrane protein (TM) YXXL signaling motifs were made. We conclude that the R3 and G4 genes and the TM YXXL motifs are not strictly required for pathogenesis but that their integrity contributes to disease frequency and latency.

Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human.

In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV), belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1). This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

Human T-cell leukemia virus type-1 Tax oncoprotein regulates G-protein signaling.

Human T-cell leukemia virus type-1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and neurological syndromes. HTLV-1 encodes the oncoprotein Tax-1, which modulates viral and cellular gene expression leading to T-cell transformation. Guanine nucleotide-binding proteins (G proteins) and G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins known and are involved in the regulation of most biological functions. Here, we report an interaction between HTLV-1 Tax oncoprotein and the G-protein beta subunit. Interestingly, though the G-protein beta subunit inhibits Tax-mediated viral transcription, Tax-1 perturbs G-protein beta subcellular localization. Functional evidence for these observations was obtained using conditional Tax-1-expressing transformed T-lymphocytes, where Tax expression correlated with activation of the SDF-1/CXCR4 axis. Our data indicated that HTLV-1 developed a strategy based on the activation of the SDF-1/CXCR4 axis in the infected cell; this could have tremendous implications for new therapeutic strategies.

Cell dynamics and immune response to BLV infection: a unifying model.

Bovine Leukemia virus (BLV) is the natural etiological agent of a lymphoproliferative disease in cattle. BLV can also be transmitted experimentally to a related ruminant species, sheep, in which the pathogenesis is more acute. Although both susceptible species develop a strong anti-viral immune response, the virus persists indefinitely throughout life, apparently at a transcriptionally silent stage, at least in a proportion of infected cells. Soon after infection, these humoral and cytotoxic activities very efficiently abolish the viral replicative cycle, permitting only mitotic expansion of provirus-carrying cells. Short term cultures of these infected cells initially indicated that viral expression protects against spontaneous apoptosis, suggesting that leukemia is a process of accumulation of long-lived cells. This conclusion was recently reconsidered following in vivo dynamic studies based on perfusions of nucleoside (bromodeoxyuridine) or fluorescent protein markers (CFSE). In sheep, the turnover rate of infected cells is increased, suggesting that a permanent clearance process is exerted by the immune system. Lymphocyte trafficking from and to the secondary lymphoid organs is a key component in the maintenance of cell homeostasis. The net outcome of the immune selective pressure is that only cells in which the virus is transcriptionally silenced survive and accumulate, ultimately leading to lymphocytosis. Activation of viral and/or cellular expression in this silent reservoir with deacetylase inhibitors causes the collapse of the proviral loads. In other words, modulation of viral expression appears to be curative in lymphocytic sheep, an approach that might also be efficient in patients infected with the related Human T-lymphotropic virus type 1. In summary, a dynamic interplay between BLV and the host immune response modulates a complex equilibrium between (i) viral expression driving (or) favoring proliferation and (ii) viral silencing preventing apoptosis. As conclusion, we propose a hypothetical model unifying all these mechanisms.

Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep.

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.

Peripheral blood B-cell death compensates for excessive proliferation in lymphoid tissues and maintains homeostasis in bovine leukemia virus-infected sheep.

The size of a lymphocyte population is primarily determined by a dynamic equilibrium between cell proliferation and death. Hence, lymphocyte recirculation between the peripheral blood and lymphoid tissues is a key determinant in the maintenance of cell homeostasis. Insights into these mechanisms can be gathered from large-animal models, where lymphatic cannulation from individual lymph nodes is possible. In this study, we assessed in vivo lymphocyte trafficking in bovine leukemia virus (BLV)-infected sheep. With a carboxyfluorescein diacetate succinimidyl ester labeling technique, we demonstrate that the dynamics of lymphocyte recirculation is unaltered but that accelerated proliferation in the lymphoid tissues is compensated for by increased death in the peripheral blood cell population. Lymphocyte homeostasis is thus maintained by biphasic kinetics in two distinct tissues, emphasizing a very dynamic process during BLV infection.

Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1.

Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV.

Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo.

Leukemogenic viruses like human T-lymphotropic virus and bovine leukemia virus (BLV) presumably persist in the host partly by latent integration of the provirus in a fraction of infected cells, leading to accumulative increase in the outgrowth of transformed cells. Furthermore, viral infection also correlates with a blockade of the apoptotic mechanisms concomitant with an apparent latency of the host cell. Conceptually, induction of viral or cellular gene expression could thus also be used as a therapeutic strategy against retroviral-associated leukemia. Here, we provide evidence that valproate, an inhibitor of deacetylases, activates BLV gene expression in transient transfection experiments and in short-term cultures of primary B-lymphocytes. In vivo, valproate injection into newly BLV-inoculated sheep did not abrogate primary infection. However, valproate treatment, in the absence of any other cytotoxic drug, was efficient for leukemia/lymphoma therapy in the sheep model leading to decreased lymphocyte numbers (respectively from 25.6, 35.7, and 46.5 x 10(3) cells per mm3 to 1.0, 10.6, and 24.3 x 10(3) cells per mm3 in three leukemic sheep) and tumor regression (from >700 cm3 to undetectable). The concept of a therapy that targets the expression of viral and cellular genes might be a promising treatment of adult T cell leukemia or tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy, diseases for which no satisfactory treatment exists so far.

Expression of S100P protein correlates with and contributes to the tumorigenic capacity of HeLa cervical carcinoma cells.

Tumor growth is associated with multiple changes at the gene expression level. Recognition of the genes differentially expressed between the cellular populations at various degrees of malignancy may provide valuable clues towards the identification of clinically useful diagnostic markers and/or therapeutic targets. In the present study, we used suppression subtractive PCR to identify differentially expressed genes with possible relevance for control of tumorigenic potential using two cervical carcinoma cell lines of the common HeLa origin, but of different capacity to generate tumors in nude mice. Screening of the subtracted libraries resulted in isolation of several known as well as novel genes including the gene encoding S100P calcium-binding protein that belongs to S100 family, whose members can bind and modulate effector proteins in a calcium-dependent manner. Expression of S100P was further studied in the context of different culture conditions and was found to correlate with the tumorigenic phenotype of the somatic cell hybrids between HeLa and normal human fibroblasts. Moreover, S100P was highly expressed in a number of tumorigenic cell lines derived from colorectal and breast carcinoma, suggesting that it is not restricted to a particular tumor type. Functional involvement of S100P in tumor growth was evaluated using tumor xenografts produced from the cells transfected with the full-length S100P cDNA. The results showed that S100P can positively affect anchorage-independent growth of the transfected cells and improve tumor formation in nude mice, suggesting that it actively participates in the control of the tumorigenic potential in vivo.

The homeobox protein MSX2 interacts with tax oncoproteins and represses their transactivation activity.

Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to immortalize primary cells in vitro. To gain insight into the molecular pathways mediating the activities of this important gene, we identified cellular proteins interacting with Tax. By means of a two-hybrid approach, we show that Tax specifically interacts with MSX2, a general repressor of gene expression. GST pull-down experiments and co-immunoprecipitation assays further confirmed binding specificity. Furthermore, the N-terminal residues 1-79 of MSX2 are required for binding, whereas the C-terminal residues 201-267 of MSX2 do not play a critical role. Whereas the oncogenic potential of Tax in primary cells was only slightly affected by overexpression of MSX2, the other function of Tax, namely LTR-dependent transcriptional activation, was inhibited by MSX2 in human HeLa and bovine B-lymphoblastoid (BL3) cell lines. This MSX2 repression function can be counteracted by overexpression of transcription factors CREB2 and RAP74. The Tax/MSX2 interplay thus results in repression of viral transcriptional activation possibly acting as a regulatory feedback loop. Importantly, this viral gene silencing is not strictly associated with a concomitant loss of Tax oncogenicity as measured by its ability to immortalize primary cells. And interestingly, MSX2 also interacts with and inhibits the transactivation function of the related Tax1 protein encoded by the Human T-cell leukemia virus type 1 (HTLV-1).