Identification and characterization of FUS/TLS as a new target of ATM

ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase), important regulators of genome stability, belong to the PIKK (phosphoinositide 3-kinase-like kinase) family of protein kinases. In the present study, DNA-affinity chromatography was used to identify DNA-binding proteins phosphorylated by these kinases. This resulted in the identification of FUS (fused in sarcoma)/TLS (translocated in liposarcoma) as an in vitro target of the PIKKs. FUS is a member of the Ewing's sarcoma family of proteins that appears to play a role in regulating genome stability, since mice lacking FUS show chromosomal instability and defects in meiosis. The residues in FUS that are phosphorylated in vitro and in vivo were identified, and phospho-specific antibodies were generated to demonstrate that FUS becomes phosphorylated at Ser(42) in vivo, primarily in response to agents that cause DSBs (double-strand breaks). DSB-induced FUS phosphorylation in vivo at Ser(42) requires ATM and not DNA-PK. Although Ser(42) is retained in the oncogenic FUS-CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein 10] fusion generated by a t(12;16)(q13;p11) chromosomal translocation, Ser(42) in FUS-CHOP is not phosphorylated after DNA damage. These results identify FUS as a new target of the ATM-signalling pathway and strengthen the notion that FUS regulates genome stability.     

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.     

BCR-ABL prevents c-jun-mediated and proteasome-dependent FUS (TLS) proteolysis through a protein kinase CbetaII-dependent pathway

The DNA binding activity of FUS (also known as TLS), a nuclear pro-oncogene involved in multiple translocations, is regulated by BCR-ABL in a protein kinase CbetaII (PKCbetaII)-dependent manner. We show here that in normal myeloid progenitor cells FUS, although not visibly ubiquitinated, undergoes proteasome-dependent degradation, whereas in BCR-ABL-expressing cells, degradation is suppressed by PKCbetaII phosphorylation. Replacement of serine 256 with the phosphomimetic aspartic acid prevents proteasome-dependent proteolysis of FUS, while the serine-256-to-alanine FUS mutant is unstable and susceptible to degradation. Ectopic expression of the phosphomimetic S256D FUS mutant in granulocyte colony-stimulating factor-treated 32Dcl3 cells induces massive apoptosis and inhibits the differentiation of the cells escaping cell death, while the degradation-prone S256A mutant has no effect on either survival or differentiation. FUS proteolysis is induced by c-Jun, is suppressed by BCR-ABL or Jun kinase 1, and does not depend on c-Jun transactivation potential, ubiquitination, or its interaction with Jun kinase 1. In addition, c-Jun-induced FUS proteasome-dependent degradation is enhanced by heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and depends on the formation of a FUS-Jun-hnRNP A1-containing complex and on lack of PKCbetaII phosphorylation at serine 256 but not on FUS ubiquitination. Thus, novel mechanisms appear to be involved in the degradation of FUS in normal myeloid cells; moreover, the ability of the BCR-ABL oncoprotein to suppress FUS degradation by the induction of posttranslational modifications might contribute to the phenotype of BCR-ABL-expressing hematopoietic cells.     

COL11A2 collagen gene transcription is differentially regulated by EWS/ERG sarcoma fusion protein and wild-type ERG

A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG and COL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG in COL11A2 gene expression, we characterized the COL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2 promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction between COL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.     

Loss of fused in sarcoma (FUS) promotes pathological Tau splicing

A subset of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients present pathological redistribution and aggregation of the nuclear protein fused in sarcoma (FUS) in the cytoplasm. Although FUS associates with the spliceosomal complex, no endogenous neuronal splicing targets have been identified. Here we identify Tau mRNA as a physiological splicing target of FUS. In mouse brain, FUS directly binds to Tau pre-mRNA, and knockdown of FUS in hippocampal neurons leads to preferential inclusion of Tau exons 3 and 10. FUS knockdown causes significant growth cone enlargement and disorganization reminiscent of Tau loss of function. These findings suggest that disturbed cytoskeletal function and enhanced expression of the neurodegeneration-associated Tau exon 10 might contribute to FTLD/ALS with FUS inclusions.     

Inhibition of anchorage-independent growth of transformed NIH3T3 cells by epithelial protein lost in neoplasm (EPLIN) requires localization of EPLIN to actin cytoskeleton

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein characterized by the presence of a single centrally located lin-11, isl-1, and mec-3 (LIM) domain. We have reported previously that EPLIN is down-regulated in transformed cells. In this study, we have investigated whether ectopic expression of EPLIN affects transformation. In untransformed NIH3T3 cells, retroviral-mediated transduction of EPLIN did not alter the cell morphology or growth. NIH3T3 cells expressing EPLIN, however, failed to form colonies when transformed by the activated Cdc42 or the chimeric nuclear oncogene EWS/Fli-1. This suppression of anchorage-independent growth was not universal because EPLIN failed to inhibit the colony formation of Ras-transformed cells. Interestingly, the localization of EPLIN to the actin cytoskeleton was maintained in the EWS/Fli-1- or Cdc42-transformed cells, but not in Ras-transformed cells where it was distributed heterogeneously in the cytoplasm. Using truncated EPLIN constructs, we demonstrated that the NH(2)-terminal region of EPLIN is necessary for both the localization of EPLIN to the actin cytoskeleton and suppression of anchorage-independent growth of EWS/Fli-1-transformed cells. The LIM domain or the COOH-terminal region of EPLIN could be deleted without affecting its cytoskeletal localization or ability to suppress anchorage-dependent growth. Our study indicates EPLIN may function in growth control by associating with and regulating the actin cytoskeleton.     

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics

Background

Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress.

Results

We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved.

Conclusions

Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.

     

Expression of human FUS/TLS in yeast leads to protein aggregation and cytotoxicity,recapitulating key features of FUS proteinopathy

Mutations in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene have been associated with amyotrophic lateral sclerosis (ALS). FUS-positive neuropathology is reported in a range of neurodegenerative diseases, including ALS and fronto-temporal lobar degeneration with ubiquitin-positive pathology (FTLD-U). To examine protein aggregation and cytotoxicity, we expressed human FUS protein in yeast. Expression of either wild type or ALS-associated R524S or P525L mutant FUS in yeast cells led to formation of aggregates and cytotoxicity, with the two ALS mutants showing increased cytotoxicity. Therefore, yeast cells expressing human FUS protein recapitulate key features of FUS-positive neurodegenerative diseases. Interestingly, a significant fraction of FUS expressing yeast cells stained by propidium iodide were without detectable protein aggregates, suggesting that membrane impairment and cellular damage caused by FUS expression may occur before protein aggregates become microscopically detectable and that aggregate formation might protect cells from FUS-mediated cytotoxicity. The N-terminus of FUS, containing the QGSY and G rich regions, is sufficient for the formation of aggregates but not cytotoxicity. The C-terminal domain, which contains a cluster of mutations, did not show aggregation or cytotoxicity. Similar to TDP-43 when expressed in yeast, FUS protein has the intrinsic property of forming aggregates in the absence of other human proteins. On the other hand, the aggregates formed by FUS are thioflavin T-positive and resistant to 0.5% sarkosyl, unlike TDP-43 when expressed in yeast cells. Furthermore, TDP-43 and FUS display distinct domain requirements in aggregate formation and cytotoxicity.     

Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that preferentially targets motor neurons. It was recently found that dominant mutations in two related RNA-binding proteins, TDP-43 (43-kDa TAR DNA-binding domain protein) and FUS/TLS (fused in sarcoma/translated in liposarcoma) cause a subset of ALS. The convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations are suggestive of a functional relationship; however, whether or not TDP-43 and FUS/TLS operate in common biochemical pathways is not known. Here we show that TDP-43 and FUS/TLS directly interact to form a complex at endogenous expression levels in mammalian cells. Binding was mediated by an unstructured TDP-43 C-terminal domain and occurred within the context of a 300–400-kDa complex that also contained C-terminal cleavage products of TDP-43 linked to neuropathology. TDP-43 C-terminal fragments were excluded from large molecular mass TDP-43 ribonucleoprotein complexes but retained FUS/TLS binding activity. The functional significance of TDP-43-FUS/TLS complexes was established by showing that RNAi silencing of either TDP-43 or FUS/TLS reduced the expression of histone deacetylase (HDAC) 6 mRNA. TDP-43 and FUS/TLS associated with HDAC6 mRNA in intact cells and in vitro, and competition experiments suggested that the proteins occupy overlapping binding sites. The combined findings demonstrate that TDP-43 and FUS/TLS form a functional complex in intact cells and suggest that convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations may reflect their participation in common biochemical processes.     

The RNA-binding Protein Fused in Sarcoma (FUS) Functions Downstream of Poly(ADP-ribose) Polymerase (PARP) in Response to DNA Damage

The list of factors that participate in the DNA damage response to maintain genomic stability has expanded significantly to include a role for proteins involved in RNA processing. Here, we provide evidence that the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS) is a novel component of the DNA damage response. We demonstrate that FUS is rapidly recruited to sites of laser-induced DNA double-strand breaks (DSBs) in a manner that requires poly(ADP-ribose) (PAR) polymerase activity, but is independent of ataxia-telangiectasia mutated kinase function. FUS recruitment is mediated by the arginine/glycine-rich domains, which interact directly with PAR. In addition, we identify a role for the prion-like domain in promoting accumulation of FUS at sites of DNA damage. Finally, depletion of FUS diminished DSB repair through both homologous recombination and nonhomologous end-joining, implicating FUS as an upstream participant in both pathways. These results identify FUS as a new factor in the immediate response to DSBs that functions downstream of PAR polymerase to preserve genomic integrity.