FUS, EWS and TAF15 are structurally similar multifunctional proteins that were first discovered upon characterization of fusion oncogenes in human sarcomas and leukemias. The proteins belong to the FET (previously TET) family of RNA-binding proteins and are implicated in central cellular processes such as regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing. In the present study, we investigated the expression and cellular localization of FET proteins in multiple human tissues and cell types.

DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders.

Members of the human FET family of RNA‐binding proteins, comprising FUS, EWSR1, and TAF15, are ubiquitously expressed and engage at several levels of gene regulation. Many sarcomas and leukemias are characterized by the expression of fusion oncogenes with FET genes as 5′ partners and alternative transcription factor‐coding genes as 3′ partners. Here, we report that the N terminus of normal FET proteins and their oncogenic fusion counterparts interact with the SWI/SNF chromatin remodeling complex. In contrast to normal FET proteins, increased fractions of FET oncoproteins bind SWI/SNF, indicating a deregulated and enhanced interaction in cancer. Forced expression of FET oncogenes caused changes of global H3K27 trimethylation levels, accompanied by altered gene expression patterns suggesting a shift in the antagonistic balance between SWI/SNF and repressive polycomb group complexes. Thus, deregulation of SWI/SNF activity could provide a unifying pathogenic mechanism for the large group of tumors caused by FET fusion oncoproteins. These results may help to develop common strategies for therapy.

The three FET (FUS, EWSR1, and TAF15) family RNA binding proteins are expressed in all tissues and almost all cell types. The disordered N‐terminal parts are always present in FET fusion oncoproteins of sarcomas and leukemia. Mutations in FUS and TAF15 cause aggregation of FET proteins in neurological disorders. Here we used recombinant proteins in pulldown experiments and mass spectrometry to identify major interaction partners of the FET N‐terminal parts. We report that FUS, EWSR1, and TAF15 form homo‐ and heterocomplexes as major binding partners and identify an evolutionarily conserved N‐terminal motif (FETBM1) that is required for this interaction. The binding is RNA and DNA independent and robust up to 1 M of NaCl. The localization of FETBM1 and its target sequences supports a simple model for FET protein aggregation as reported in neurological disorders such as amyotrophic lateral sclerosis, frontotemporal dementia, and essential tremor. The FETBM1 localization also explains the binding of normal full‐length FET proteins to their oncogenic fusion proteins.—Thomsen, C., Grundevik, P., Elias, P., Ståhlberg, A., Åman, P., A conserved N‐terminal motif is required for complex formation between FUS, EWSR1, TAF15 and their oncogenic fusion proteins. FASEB J. 27, 4965–4974 (2013). www.fasebj.org

Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.