Oncogene database

2521

FUS

FUS RNA binding protein;FUS;FUS RNA binding protein

75 kDa DNA-pairing protein|RNA-binding protein FUS|fus-like protein|fused in sarcoma|fusion gene in myxoid liposarcoma|heterogeneous nuclear ribonucleoprotein P2|oncogene FUS|oncogene TLS|translocated in liposarcoma protein

16p11.2

protein-coding

We have previously developed an assay to measure DNA homologous pairing activities in crude extracts: The POM blot. In mammalian nuclear extracts, we detected two major DNA homologous pairing activities: POMp100 and POMp75. Here, we present the purification and identification of POMp75 as the pro-oncoprotein TLS/FUS. Because of the pro-oncogene status of TLS/FUS, we studied in addition, the relationships between cell proliferation and POM activities. We show that transformation of human fibroblasts by SV40 large T antigen results in a strong increase of both POMpl00 and TLS/POMp75 activities. Although detectable levels of both POMp100 and TLS/POMp75 are observed in non-immortalized fibroblasts or lymphocytes, fibroblasts at mid confluence or lymphocytes stimulated by phytohaemaglutinin, show higher levels of POM activities. Moreover, induction of differentiation of mouse F9 line by retinoic acid leads to the inhibition of both POMp100 and TLS/POMp75 activities. Comparison of POM activity of TLS/FUS with the amount of TLS protein detected by Western blot, suggests that the POM activity could be regulated by post-translation modification. Taken together, these results indicate that POMp100 and TLS/POMp75 activities are present in normal cells but are connected to cell proliferation. Possible relationship between cell proliferation, response to DNA damage and DNA homologous pairing activity of the pro-oncoprotein TLS/FUS are discussed.

The evolutionarily conserved BTB/POZ domain from the promyelocytic leukemia zinc finger (PLZF) oncoprotein mediates transcriptional repression through the recruitment of corepressor proteins containing histone deacetylases in acute promyelocytic leukemia. We have determined the 2.0 A crystal structure of the BTB/POZ domain from PLZF (PLZF-BTB/POZ), and have carried out biochemical analysis of PLZF-BTB/POZ harboring site-directed mutations to probe structure-function relationships. The structure reveals a novel alpha/beta homodimeric fold in which dimer interactions occur along two surfaces of the protein subunits. The conservation of BTB/POZ domain residues at the core of the protomers and at the dimer interface implies an analogous fold and dimerization mode for BTB/POZ domains from otherwise functionally unrelated proteins. Unexpectedly, the BTB/POZ domain forms dimer-dimer interactions in the crystals, suggesting a mode for higher-order protein oligomerization for BTB/POZ-mediated transcriptional repression. Biochemical characterization of PLZF-BTB/POZ harboring mutations in conserved residues involved in protein dimerization reveals that the integrity of the dimer interface is exquisitely sensitive to mutation and that dimer formation is required for wild-type levels of transcriptional repression. Interestingly, similar mutational analysis of residues within a pronounced protein cleft along the dimer interface, which had been implicated previously for interaction with corepressors, has negligible effects on dimerization or transcriptional repression. Together, these studies form a structure-function framework for understanding BTB/POZ-mediated oligomerization and transcriptional repression properties.

Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.

In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor kappaB (NF-kappaB) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-kappaB-mediated transactivation induced by physiological stimuli such as tumor necrosis factor alpha, interleukin-1beta, and overexpression of NF-kappaB-inducing kinase. TLS augmented NF-kappaB-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-beta gene. These results suggest that TLS acts as a coactivator of NF-kappaB and plays a pivotal role in the NF-kappaB-mediated transactivation.

TLS/FUS is a nucleic acid-binding protein whose N-terminal half functions as a transcriptional activator domain in fusion oncoproteins found in human leukemias and liposarcomas. Previous reports have suggested a role for TLS/FUS in transcription and splicing processes. Here we report the association of TLS/FUS with the nuclear matrix and investigate its role in splicing. Splicing of two pre-mRNAs was inhibited in a TLS/FUS-immunodepleted extract and could only be partly restored by addition of recombinant TLS/FUS or/and SR proteins, known interaction partners of TLS/FUS. The subsequent analysis of TLS/FUS immunoprecipitates revealed that, in addition to the SR proteins SC35 and SRp75, the splicing factor PTB (hnRNPI) and the splicing coactivator SRm160 are complexed with TLS/FUS, thus explaining the inability to restore splicing completely. Coimmunolocalization confirmed the nuclear matrix association and interaction of TLS/FUS with PTB, SR proteins, and SRm160. Our results suggest that the matrix protein TLS/FUS plays a role in spliceosome assembly.

The myxoid/round cell liposarcoma oncogene FUS-DDIT3 is the result of a translocation derived gene fusion between the splicing factor FUS and DDIT3. In order to investigate the downstream targets of DDIT3, and the transforming effects of the FUS-DDIT3 fusion protein, we have introduced DDIT3-GFP and FUS-DDIT3-GFP constructs into a human fibrosarcoma cell line. The gene expression profiles of stable transfectants were compared to the original fibrosarcoma cell line by microarray analysis. We here report that the NFkappaB and C/EBP beta controlled gene IL6 is upregulated in DDIT3- and FUS-DDIT3-expressing fibrosarcoma cell lines and in myxoid liposarcoma cell lines. Strong expression of the tumor associated multifunctional cytokine interleukin 6 was confirmed both at mRNA and protein level. Knockdown experiments using siRNA against CEBPB transcripts showed that the effect of FUS-DDIT3 on IL6 expression is C/EBP beta dependent. Chromatin immunoprecipitation revealed direct interaction between the IL6 promoter and the C/EBP beta protein. In addition, the effect of DDIT3 and FUS-DDIT3 on the expression of other acute phase genes was examined using real-time PCR. We demonstrate for the first time that DDIT3 and FUS-DDIT3 show opposite transcriptional regulation of IL8 and suggest that FUS-DDIT3 may affect the synergistic activation of promoters regulated by C/EBP beta and NFkappaB.

The oncogenic TLS-ERG fusion protein is found in human myeloid leukemia and Ewing s sarcoma as a result of specific chromosomal translocation. To unveil the potential mechanism(s) underlying cellular transformation, we have investigated the effects of TLS-ERG on both gene transcription and RNA splicing. Here we show that the TLS protein forms complexes with RNA polymerase II (Pol II) and the serine-arginine family of splicing factors in vivo. Deletion analysis of TLS-ERG in both mouse L-G myeloid progenitor cells and NIH 3T3 fibroblasts revealed that the RNA Pol II-interacting domain of TLS-ERG resides within the first 173 amino acids. While TLS-ERG repressed expression of the luciferase reporter gene driven by glycoprotein IX promoter in L-G cells but not in NIH 3T3 cells, the fusion protein was able to affect splicing of the E1A reporter in NIH 3T3 cells but not in L-G cells. To identify potential target genes of TLS-ERG, the fusion protein and its mutants were stably expressed in both L-G and NIH 3T3 cells through retroviral transduction. Microarray analysis of RNA samples from these cells showed that TLS-ERG activates two different sets of genes sharing little similarity in the two cell lines. Taken together, these results suggest that the oncogenic TLS-ERG fusion protein transforms hematopoietic cells and fibroblasts via different pathways.

BACKGROUND & AIMS: beta-Catenin is a downstream effector of the Wnt signaling pathway and is believed to exert its oncogenic function by activating T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcriptional factors. However, it is still uncertain whether the diverse effects of beta-catenin are caused solely by aberrant gene transactivation. In this study, we used a proteomics approach to obtain further insight into the functional properties of nuclear beta-catenin. METHODS: The protein assembly of a native beta-catenin-containing complex in nuclear extracts from a colorectal cancer cell line, DLD-1, was identified using immunoprecipitation and mass spectrometry. RESULTS: beta-Catenin physically interacted with fusion (FUS)/translocated in liposarcoma (TLS) and various RNA-binding proteins. The expression of FUS/TLS was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. The transient transfection of FUS suppressed beta-catenin-evoked gene transactivation of TCF/LEF, and beta-catenin transfection affected the splicing pattern of the E1A minigene and induced a novel splicing variant of estrogen receptor (ER)-beta exerting a dominant-negative activity. CONCLUSIONS: Human cancer expresses a large variety of alternatively spliced messenger RNA (mRNA), but the precise molecular mechanisms responsible for cancer-related alternative splicing are largely unknown. In this study, we demonstrated the interaction of beta-catenin with FUS/TLS and other RNA-binding proteins involved in the regulation of pre-mRNA splicing. Certain mRNA splicing abbreviations seen in human cancers may be induced by the activation of the Wnt signaling pathway.

Myxoid/round cell liposarcoma (MLS/RCLS) is the most common subtype of liposarcoma. Most MLS/RCLS carry a t(12;16) translocation, resulting in a FUS-DDIT3 fusion gene. We investigated the role of the FUS-DDIT3 fusion in the development of MLS/RCLS in FUS-DDIT3- and DDIT3-transfected human HT1080 sarcoma cells. Cells expressing FUS-DDIT3 and DDIT3 grew as liposarcomas in severe combined immunodeficient mice and exhibited a capillary network morphology that was similar to networks of MLS/RCLS. Microarray-based comparison of HT1080, the transfected cells, and an MLS/RCLS-derived cell line showed that the FUS-DDIT3- and DDIT3-transfected variants shifted toward an MLS/RCLS-like expression pattern. DDIT3-transfected cells responded in vitro to adipogenic factors by accumulation of fat and transformation to a lipoblast-like morphology. In conclusion, because the fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype when expressed in a primitive sarcoma cell line, MLS/RCLS may develop from cell types other than preadipocytes. This may explain the preferential occurrence of MLS/RCLS in nonadipose tissues. In addition, development of lipoblasts and the typical MLS/RCLS capillary network could be an effect of the DDIT3 transcription factor partner of the fusion oncogene.

FUS-DDIT3 is a chimeric oncogene generated by the most common chromosomal translocation t(12;16)(q13;p11) associated to liposarcomas. The application of transgenic methods and the use of primary mesenchymal progenitor cells to the study of this sarcoma-associated FUS-DDIT3 gene fusion have provided insights into their in vivo functions and suggested mechanisms by which lineage selection may be achieved. These studies indicate that FUS-DDIT3 contributes to differentiation arrest acting at a point in the adipocyte differentiation process after induction of peroxisome proliferator-activated receptor gamma (PPARgamma) expression. To test this idea within a living mouse, we generated mice expressing FUS-DDIT3 within aP2-positive cells, because aP2 is a downstream target of PPARgamma expressed at the immature adipocyte stage. Here, we report that FUS-DDIT3 expression was successfully induced at the aP2 stage of differentiation both in vivo and in vitro. aP2-FUS-DDIT3 mice do not develop liposarcomas and exhibit an increase in white adipose tissue size. Consistent with in vivo data, mouse embryonic fibroblasts (MEFs) obtained from aP2-FUS-DDIT3 mice not only were capable of terminal differentiation but also showed an increased capacity for adipogenesis in vitro compared with wild-type MEFs. Taken together, this study provides genetic evidence that the presence of FUS-DDIT3 in an aP2-positive cell is not enough to cause liposarcoma development and establishes that PPARgamma inactivation is required for liposarcoma development.

The FUS-CHOP fusion protein has been found to be instrumental for specific oncogenic processes in liposarcoma, but its ability to induce metastasis and the underlying mechanisms by which this can be achieved remain unknown. To dissect its functional role in this context, we stably overexpressed this protein in SW872 liposarcoma and HT1080 fibrosarcoma cell lines, and were able to demonstrate that forced expression of FUS-CHOP significantly increases migration and invasion, as well as enhances lung and liver metastasis in the in vivo chicken chorioallantoic membrane (CAM) model, that is proliferation independent. Additionally, FUS-CHOP enhances the expression of matrix-metalloproteinases -2 and -9, and transactivates their promoters in vitro. mutational analysis showed that C/EBP-beta- (-769/-755), NF-kappaB (-525/-516) and CREB/AP-1 (-218/-207) sites were important for MMP-2 and NF-kappaB (-604/-598), AP-1 (-539/-532) and AP-1 (-81/-72) for MMP-9 transactivation. Moreover, a direct in vivo interaction of FUS-CHOP was observed in case of the MMP-2 promoter within region (-769/-207). siRNA data revealed that MMP-2 expression is essential in the FUS-CHOP induced metastatic phenotype. MMP-2-mRNA and protein expression correlated significantly with FUS-CHOP positivity in 46 resected patient liposarcoma tissues. We have for the first time provided substantial evidence for the FUS-CHOP oncoprotein as an inducer of metastasis that is due to the transcriptional induction of specific tumor-associated proteases. Insights gained from this study not only support a deeper understanding of the mechanistic properties of FUS-CHOP, but also open up new avenues for targeted therapy.

Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBPbeta in naive NIH3T3 cells, and their ability to inhibit C/EBPbeta-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBPbeta and inhibit C/EBPbeta DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP s putative transactivation activity. Another adipogenic transcription factor, PPARgamma2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBPbeta from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process.

The gene encoding the human TLS protein, also termed FUS, is located at the site of chromosomal translocations in human leukemias and sarcomas where it forms a chimeric fusion gene with one of several different genes. To identify interacting partners of TLS, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the C-terminal region of TLS in the bait plasmid. Two cDNAs encoding members of the serine-arginine (SR) family of proteins were isolated. The first SR protein is the mouse homolog of human splicing factor SC35, and the second SR member is a novel 183-amino acid protein that we term TASR (TLS-associated serine-arginine protein). cDNA cloning of human TASR indicated that mouse and human TASR have identical amino acid sequences. The interactions between TLS and these two SR proteins were confirmed by co-transfection and immunoprecipitation studies. In vivo splicing assays indicated that SC35 and TASR influence splice site selection of adenovirus E1A pre-mRNA. TLS may recruit SR splicing factors to specific target genes through interaction with its C-terminal region, and chromosomal translocations that truncate the C-terminal region of TLS may prevent this interaction. Thus TLS translocations may alter RNA processing and play a role in malignant transformation.