Ewing's family tumors (EFTs) are highly malignant tumors arising from bone and soft tissues that exhibit EWS-FLI1 or variant EWS-ETS gene fusions in more than 85% of the cases. Here we show that CIC, a human homolog of Drosophila capicua which encodes a high mobility group box transcription factor, is fused to a double homeodomain gene DUX4 as a result of a recurrent chromosomal translocation t(4;19)(q35;q13). This translocation was seen in two cases of soft tissue sarcoma diagnosed as Ewing-like sarcoma. CIC-DUX4 exhibits a transforming potential for NIH 3T3 fibroblasts, and as a consequence of fusion with a C-terminal fragment of DUX4, CIC acquires an enhanced transcriptional activity, suggesting that expression of its downstream targets might be deregulated. Gene expression analysis identified the ETS family genes, ERM/ETV5 and ETV1, as potential targets for the gene product of CIC-DUX4. Indeed, CIC-DUX4 directly binds the ERM promoter by recognizing a novel target sequence and significantly up-regulates its expression. This study clarifies the function of CIC and its role in tumorigenesis, as well as the importance of the PEA3 subclass of ETS family proteins in the development of EFTs arising through mechanisms different from those involving EWS-ETS chimeras. Moreover, the study identifies the role of DUX4 that is closely linked to facioscapulohumeral muscular dystrophy in transcriptional regulation.

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, ␤-transducin repeat-containing protein (␤TrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a ␤TrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes. Viral protein U (Vpu)2 is an 81-amino acid type I integral membrane phosphoprotein expressed by human immunodeficiency virus type 1 (HIV-1) (1, 2) and several simian immunodeficiency viruses (3-6). Vpu is not incorporated into virus particles (7), indicating that it acts exclusively in virus-producer cells. Indeed, Vpu is known to play two distinct roles during the later stages of infection. First, Vpu interacts with newly synthesized CD4 molecules complexed with the gp160 envelope glycoprotein precursor in the endoplasmic reticulum (8, 9) and recruits the ␤-transducin repeat-containing protein 1 (␤TrCP-1) subunit of the Skp1-Cullin1-F-box ubiquitin ligase complex (10) as well as ␤TrCP-2 (11) through its phosphoserine residues at positions 52 and 56 in the cytoplasmic (CT) domain (12,13). This event results in proteasome-mediated degradation of CD4 (10, 14, 15) allowing gp160 to resume transport toward the cell surface for virion incorporation. Second, Vpu mediates the enhancement of virion release (16 -18) in a cell type-dependent manner (e.g. HeLa cells require Vpu, whereas COS7 cells do not (19,20)), and its absence leads to the accumulation of viral particles at the cell surface (21).In contrast to the effect of Vpu on CD4 degradation, little had been known about the mechanism by which Vpu enhances the release of virions. The finding that HeLa-COS7 heterokaryons exhibited HeLa-type properties suggested that Vpu-responsive HeLa cells might harbor endogenous a restriction factor(s) that could be counteracted by this viral protein (22), as seen in Vifresponsive cells harboring the antiretroviral factor APOBEC3G counteracted by Vif (23). Neil et al. (24) showed that Vpu-deficient viral particles accumulated ...

IntroductionCarcinogenesis is a consecutive process of multiple genetic and epigenetic alterations that affect the target cell to acquire growth advantages and dysregulated growth from tissue environment. 1 Plenty of genetic mutations have been discovered in both human and animal cancers. It should be noted that the mutations are cell-type specific and that there is specific genetic cooperation in the multi-step evolution of premalignant cells. The combination of genetic mutations alters cell fate, and specific genetic cooperation is therefore important for tumor phenotypes and biologic behaviors of cancer cells.Proper expression of AbdB-like Hox genes is a key molecular process in hematopoiesis and their deregulation perturbs myeloid differentiation, eventually resulting in leukemia with several cooperative genetic events. 2 One of the important Hox genes in myeloid leukemogenesis is Hoxa9, which has been identified as a target for ecotropic retroviral integration in BXH2 murine AML 3 as well as a fusion partner of NUP98 in human AML with t(7;11)(p15;p15) translocation. 4,5 Importantly, cooperative activation of the Hox cofactor Meis1 is important for both Hoxa9-and NUP98-HOXA9-induced transformation of murine bone marrow cells. 6,7 Recent studies have suggested that Meis1 might activate Flt3 receptor tyrosine kinase, which plays an important role in myeloid leukemogenesis. [8][9][10] However, it remains to be clarified what kinds of subsequent molecular processes are required for complete leukemogenesis, and it is important to understand subsequent molecular steps that are cooperative for Hoxa9 and Meis1 activation.We have addressed this issue by using retroviral insertional mutagenesis that has been applied for identification of oncogenes and tumor suppressor genes. 11 Plenty of important disease genes have been isolated by this technique and many of the genes are in fact involved in human cancers. Retroviral integration in the bone marrow cells is randomly distributed over the whole genome, and the cells with integrations that contribute growth advantages are subsequently selected and clonally expanded. Cooperative genetic effects in leukemogenesis can be achieved by multi-copy integration of retroviruses. The multiple integrations do not occur simultaneously, but it is likely that the process is consecutive, since such cooperative integrations are extremely rare without any predisposition. When there is a primary genetic alteration in the target cell, genetic cooperation for the first hit can be investigated by this technique. 12 Retrovirus-mediated gene transfer and bone marrow transfer of Hoxa9 and Meis1 induce AML at 100% penetrance. 6,13 In this study we have found that there is clonal selection of the transduced bone marrow cells at the transformation step, and we have systematically cloned retroviral integration sites in Hoxa9/Meis1-induced AML. Inverse polymerase chain reaction (PCR) experiments identified 6 common integration sites (CISs), and candidate cooperative genes for Hoxa9/Meis1 coactivation...

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the transGolgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER. Entry of proteins into the secretory pathway via the rough endoplasmic reticulum (ER)3 is essential for intracellular transport of proteins to secretory compartments within the cell and finally to the extracellular milieu. In tissues with high secretory activity, such as the pancreas, the secretory apparatus is highly developed. One of the morphological hallmarks in such tissues is drastic proliferation of rough ER membranes that are densely occupied by ribosomes, whereas the rough ER in other tissues forms a loose network of tubular cisternae sparsely studded with ribosomes (1). However, the molecular basis for the biogenesis and proliferation of the rough ER in secretory tissues is largely unknown (1, 2).Collagen is one of the major components of the extracellular matrix (ECM) in connective tissues such as skin, tendon, and bone. It is synthesized on the ER membrane as a precursor form, i.e. procollagen, and secreted by professional secretory cells including fibroblasts. Fully consistent with the normal secretory pathway, procollagen is cotranslationally translocated into the lumen of the ER. Much interest has been focused on the mechanisms of its folding and trimerization processes in the ER, such as the hydroxylation enzymes for proline and lysine residues (reviewed in Refs. 3 and 4)). Recently, there has been considerable interest in the intracellular trafficking mechanism of procollagen as a representative model for supramolecular cargos (5-7). In addition, the regulation of procollagen biosynthesis has been intensively studied at the transcriptional level (8, 9) as well as at the...