Molecular genetics of chromosome translocations involving EWS and related family members

JUNGHO KIM1 and JERRY PELLETIER1,2

1 Department of Biochemistry
2 Department of Oncology, McGill University, Montreal, Quebec, Canada H3G 1Y6


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 TRANSLOCATIONS INVOLVING THE EWS...
 CONCLUSIONS
 REFERENCES
 
Kim, Jungho, and Jerry Pelletier. Molecular genetics of chromosome translocations involving EWS and related family members. Physiol. Genomics 1: 127–138, 1999.—Many types of sarcomas are characterized by specific chromosomal translocations that appear to result in the production of novel, tumor-specific chimeric transcription factors. Many of these show striking similarities: the emerging picture is that the amino-terminal domain of the fusion product is donated by the Ewing's sarcoma gene (EWS) or a related member from the same gene family, whereas the carboxy-terminal domain often consists of a DNA-binding domain derived from one of a number of transcription factors. Given the observation that the different translocation partners of the EWS protooncogene are associated with distinct types of sarcomas, the functional consequence of fusing EWS (or a related family member) to a different DNA-binding domain can only be understood in the context of functional studies that define the specificity of action of the different fusion products. An understanding of the molecular structure and function of these translocations provides new methods for diagnosis and novel targets for therapeutics.

EWS; oncogene; transformation; cancer genetics


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 TRANSLOCATIONS INVOLVING THE EWS...
 CONCLUSIONS
 REFERENCES
 
AN UNDERSTANDING of the physiological consequences of genetic changes in cellular protooncogenes and tumor-suppressor genes for progression of the neoplastic phenotype would enable the research community to identify novel targets against which new therapies may be considered. For a large, effective therapeutic index to be achieved, the most desired scenario is one in which the target is unique to the cancer cell. Such is the case with the product of translocation fusions that are associated with a large number of sarcomas and that have acquired novel biochemical properties.

Three genetic mechanisms are known to activate oncogenes in human neoplasms. 1) Various types of mutations, such as base substitutions, deletions, and inversions, are capable of activating protooncogenes. 2) Gene amplification results in expansion of gene copy number, a process that occurs through redundant replication of genomic DNA and often gives rise to karyotypic abnormalities such as double-minute chromosomes and homogeneous staining regions. 3) Recurring chromosomal rearrangements are often detected in some solid tumors, as well as in hematological malignancies (79). These rearrangements consist mainly of chromosomal translocations and, less frequently, chromosomal inversions. Chromosomal rearrangements are thought to lead to neoplastic transformation by at least two different mechanisms: the transcriptional activation of protooncogenes or the creation of novel gene fusion products. In the latter situation, the resulting juxtaposition of segments from two different genes gives rise to a composite structure consisting of the amino-terminal domain (NTD) of one gene and the carboxy-terminal domain (CTD) of another gene. The emerging picture in many sarcomas is that the NTD of the fusion product is donated by the Ewing's sarcoma gene (EWS) or a related member from the same gene family, whereas the CTD often consists of a DNA-binding domain derived from a transcription factor (10, 23, 94, 113, 114). The purpose of this review is to present the current state of knowledge of chromosomal translocations in sarcomas involving the EWS protooncogene and its related family members. Given the observation that the different translocation partners of the EWS protooncogene are associated with distinct types of sarcomas, the functional consequence of fusing EWS (or a related family member) to a different DNA-binding domain can only be understood in the context of functional studies that define the specificity of action of the different fusion products.


    TRANSLOCATIONS INVOLVING THE EWS GENE
 TOP
 ABSTRACT
 INTRODUCTION
 TRANSLOCATIONS INVOLVING THE EWS...
 CONCLUSIONS
 REFERENCES
 
Ewing's Sarcoma/Primitive Neuroectodermal Tumor
The Ewing's sarcoma protooncogene, EWS.
The general structure of the EWS protooncogene is outlined in Fig. 1. The EWS gene contains an open reading frame of 1,968 bp and encodes a protein of 656 amino acids. It spans a 40-kbp region on chromosome 22, is composed of 17 exons interrupted by 16 introns, and is oriented centromere to telomere on chromosome 22q12. Alternative splicing of the EWS gene produces two EWS transcripts (EWS and EWS-b) that differ with respect to the presence or absence of exons 8 and 9 (67) (Fig. 1). On the basis of homology features of the primary amino acid sequence, the EWS coding region can be divided into different domains. The first seven exons encode the NTD of EWS and consist of 285 amino acid residues containing a degenerate repeated motif with a frequently occurring Ser-Tyr dipeptide. This region is also rich in glutamine, threonine, and proline residues. The protein region between amino acids 157 and 262 shares 40% homology with the CTD of the large subunit of eukaryotic RNA polymerase II protein (CTD-pol II) (19).



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Fig. 1. Schematic representation of chromosomal localization and genomic organization of the EWS gene and the structureal features of the EWS protooncoprotein. The EWS gene spans a region of ~40-kb on chromosome 22 and contains 17 exons interrupted by 16 introns. The chromosomal breakpoint is indicated by an arrowhead. Restriction mapping of the rearrangements and analysis of the structure of the chimeric RNAs indicate that ~90% of EWS breakpoints occur within introns 7 and 8, in approximately equal proportions (76, 114). Interestingly, breakpoints in either of these introns result in chimeric RNAs that include exon 7 but not exon 8 of EWS because exon 8 appears to be systematically spliced out of the chimeric RNAs (114). As a result of an alternative splicing event that results in either inclusion or exclusion of exons 8 and 9, the EWS gene encodes two different transcripts: 1) EWS, which encodes a protein of 656 amino acids, and 2) EWS-b, which encodes a variant EWS polypeptide of 583 amino acids. The activation domain of EWS protein is represented by a shaded box. The three glycine-, arginine-, and proline-rich (GRP-rich) regions and RNA-binding domain are delineated. CTD, carboxy-terminal domain.

 
Exons 11–13 encode a putative 85-amino acid RNA-binding domain, termed the RNA recognition motif (RRM) (33, 43), present in several RNA-binding proteins. Three regions containing glycine-, arginine-, and proline-rich (i.e., GRP-rich) motifs are mainly encoded by exons 8–9, 14, and 16. These are similar to tracts that have been shown to bind RNA in other proteins and in various single-stranded nucleic acid-binding proteins, including SSB1 protein, nucleolin, fibrillarin, hnRNPs, and NOP1 (44). Moreover, the carboxy-terminal GRP motif of hnRNP A1, which shares 43% homology with the third and longest GRP motif of EWS, possesses strand-annealing activity and is capable of binding to both RNA and single-stranded DNA (48, 49). Taken together, these results indicate that the carboxy-terminal portion of EWS is involved in RNA recognition. Consistent with this interpretation, EWS can bind to poly(G) and poly(U), and the conserved GRP box present in the extreme carboxy-terminal region of EWS can function as an RNA-binding domain (67). In the absence of appropriate controls for specificity in these experiments, the physiological function of the EWS RRM is still an open question. Interestingly, a difference in RNA binding between the two EWS isoforms has been noticed: EWS can bind both poly(G) and poly(U), whereas EWS-b only recognizes poly(G) (67).

It has recently been reported that ZFM1 (zinc finger gene in the MEN 1 locus), identified as a downstream effector of p53-induced apoptosis (1), interacts with EWS in yeast two-hybrid assays and pull-down experiments (110). ZFM1 encodes a nuclear protein with a hnRNP KH homology domain (RNA-binding motif) and a zinc knuckle motif, both implicated in nucleic acid binding. The interacting region maps to a 37-amino acid domain within the NTD of EWS. Overexpression of ZFM1 in HepG2 cells repressed the transactivation of a reporter gene by EWS, and this repression correlated with the ability of ZFM1 to bind EWS. Interestingly, two EWS-related proteins, TLS/FUS (21) and hTAFII68 (5), also interact with ZFM1 (110).

EWS contains an IQ domain (Fig. 1) that is phosphorylated by protein kinase C (PKC) and interacts with calmodulin (CaM) (24). The IQ domain consists of 20 amino acids that serve as a general regulatory domain in proteins involved in CaM binding and PKC phosphorylation (29). PKC phosphorylation of EWS inhibits its binding to RNA homopolymers, and, conversely, RNA binding to EWS interferes with PKC phosphorylation (24).

The DNA sequence in the 5' region of the EWS gene has features of a CpG-rich island and lacks canonical promoter elements, such as TATA and CCAAT consensus sequences (76). The 600-bp sequence immediately upstream of the ATG codon contains 67% G+C and encompasses the first exon and extends into the first intron. Sequence analysis of the 5' end of EWS cDNAs suggests that the transcription of the EWS gene initiates at multiple sites. Multiple transcription start sites associated with an absence of TATA sequence and a high incidence of unmethylated CpG dinucleotides are recognized features of the promoter region for many housekeeping genes (9). These characteristics of the EWS promoter region may provide the basis for the widespread expression of the EWS gene (114).

The ETS family of transcription factors.
Ewing's sarcoma is a highly malignant, small-round-cell sarcoma that occurs mainly in children and young adults and accounts for ~30% of primary bone tumors in this age group (99). Although lacking any morphological characteristics in many cases, Ewing's sarcoma cells sometimes exhibit neural differentiation (57). In this sense, Ewing's sarcoma cells resemble primitive neuroectodermal tumor (PNET) cells. Increasing evidence suggests that these tumors have a common neural-crest origin but may exhibit variable neural differentiation and tissue localization. Ewing's sarcoma/PNET tumors are mostly characterized by a recurrent t(11;22)(q24;q12) chromosomal translocation (114). This translocation fuses the 5' end of the EWS gene located on chromosome 22 to the 3' end of the Fli-1 gene, a member of the ETS gene family, on chromosome 11 (Table 1) (114). Recently, other types of Ewing's sarcoma translocations have been identified, in which ERG (94, 114), ETV1 (40), E1A-F (42), and FEV (74) are involved instead of the Fli-1 gene (Table 1). The common thread here is that the EWS NTD is fused to the DNA-binding domain of an ETS oncogene superfamily member of transcription factors (4, 78, 85). The ETS family comprises proteins that share a unique DNA-binding domain, the ETS domain (reviewed in Ref. 20). Based on the homology within the ETS domain, ETS proteins are divided into different subfamilies. Most ETS members bind to DNA as monomeric proteins. The ETS DNA-binding domain forms a winged helix-turn-helix motif that allows ETS proteins to interact with an ~10-bp-long GGAA/T-containing DNA element (75, 106). It has been shown that Fli-1 and ERG proteins bind to DNA in a sequence-specific manner and function as transcriptional activators (85), but their function in normal cells remains unknown.


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Table 1. Tumor-specific chromosomal translocations involving EWS or related family members

 
EWS/Fli-1.
In 85% of Ewing's sarcoma/PNET cases, the ETS domain is derived from Fli-1, creating an EWS/Fli-1 chimera as a result of a t(11;22)(q24;q12) translocation. There are at least nine types of chimeric EWS/Fli-1 transcripts that have been detected, representing different combinations of exons from EWS and Fli-1. The two most common ones are designated type I and II and account for ~80% of all cases with chimeric EWS/Fli-1 transcripts (Fig. 2). The resultant EWS/Fli-1 fusion protein (type I) is a stronger transcriptional activator than Fli-1 (2, 56, 61, 63, 64, 68). EWS/Fli-1 displays the same DNA-binding specificity and affinity as Fli-1 (61). Overexpression of EWS/Fli-1 in NIH/3T3 cells results in oncogenic transformation, in contrast to Fli-1, which has no transforming potential in this assay (11, 56, 63, 64, 69). Both EWS and Fli-1 sequences are necessary for the transforming activity of this chimeric oncogene (63). Transformation by EWS/Fli-1 is thought to disrupt normal growth and differentiation either by more efficiently activating the Fli-1 target genes or by inappropriately modulating genes normally unresponsive to the Fli-1 gene.



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Fig. 2. Schematic diagram of EWS/Fli-1 chimeras generated by fusing the EWS and Fli-1 genes. In the two types of chimeric proteins, the carboxy-terminal portion of EWS containing the putative RNA-binding domain is replaced by the carboxy-terminal part of Fli-1 containing the ETS DNA-binding domain. The EWS NTD is shown as a graded shaded box. The ETS domain is represented, and the differences between type I and type II EWS/Fli-1 fusion products are illustrated. The type II Ewing's sarcoma/Fli-1 protein contains the insertion of 22 additional amino acids from Fli-1. Positions of the interrupted codons are indicated in each case.

 
Although the genes specifically regulated by EWS/ETS fusions remain largely unknown, recent studies have identified a few candidate targets of EWS/Fli-1 (11, 62). These include c-myc (2), Stromelysin (11), EAT-2 (EWS/Fli-1-activated transcript 2) (98), and manic fringe (62), all genes upregulated by EWS/Fli-1. Interestingly, overexpression of manic fringe in NIH/3T3 cells renders them tumorigenic (62).

EWS/ERG-1.
In ~10% of Ewing's sarcoma tumors, EWS is fused to the DNA-binding domain of the ERG gene via a t(21;22)(q22;q12) chromosome translocation (94). Human EGR-1 and Fli-1 display 98% identity (83 of 85 amino acids) in their DNA-binding domains, while differing significantly in the rest of the proteins (overall identity, 68%). It is therefore possible that they bind to the same target sequences but diverge in tissue-specific function. The EWS/ERG protein also encodes a transcriptional activator. Aberrant EWS/ERG protein shows altered DNA-binding and stronger transcriptional activation properties compared with the normal ERG protein (67).

EWS/ETV1.
A variant t(7;22)(p22;q12) in Ewing's sarcoma has been previously reported (95), fusing EWS to ETV1 (40). As a result of this tumor-specific rearrangement, EWS/ETV1 transcripts are expressed exclusively in t(7;22)-containing tumor cells, and the fusion protein has sequence-specific DNA-binding activity (40). ETV1 is the human homolog of the murine ETS gene ER81, which was isolated using degenerate oligonucleotides designed to recognize conserved regions within the DNA-binding domain of ETS-related proteins (14). The involvement of ETV1 in Ewing's sarcoma could not be predicted based on homology with either Fli-1 or ERG-1. Unlike these two ETS family members, ETV1 has only 58% and 63% amino acid identity with the Fli-1 and ERG-1 ETS domains, respectively (40). ETV1 is more closely related to a distinct subfamily of ETS genes, whose other members, E1A-F, have also been implicated in tumorigenesis.

EWS/E1A-F.
EWS/E1A-F is a newly recognized chimeric gene resulting from fusion of the transactivation domain of EWS with E1A-F [encoding the adenovirus E1A enhancer-binding proteins (35) detected in one case of Ewing's sarcoma (102)]. Sequencing of the amplified chimeric cDNA product revealed that exon 7 of EWS is fused to the 3' DNA-binding domain of the E1A-F gene as a result of a t(17;22)(q21;q12) translocation (35). The E1A-F gene was isolated by virtue of its ability to bind to adenovirus E1A enhancer sites (35). The E1A-F protein contains an ETS domain and is thought to be the human homolog of mouse PEA3, a gene frequently overexpressed in metastatic mammary adenocarcinoma (100). E1A plays an important role in cell growth, and E1A-F activates the transcription of matrix metalloproteinase genes that are associated with invasion and metastasis of tumor cells (35). This latter point is quite interesting given that Ewing's sarcoma is a highly invasive and metastatic cancer.

EWS/FEV.
Recently, a new member of the ETS family, called FEV (fifth Ewing variant), has been found to be fused to EWS (Table 1). The FEV gene is encoded by 3 exons on chromosome 2. It encodes a 238-amino acid protein that contains an ETS DNA-binding domain closely related to that of Fli-1 and ERG. However, the NTD of FEV is only 42 amino acids long, suggesting that FEV is lacking important transcription regulatory domains contained in Fli-1 and ERG NTDs. The CTD of FEV is characterized by a 112-amino acid domain rich in alanine, proline, leucine, and glycine residues and includes a continuous stretch of 12 alanines. These features are sometimes observed in transcriptional repressors, suggesting that FEV may repress transcription (74). It remains to be established if fusion of FEV to EWS converts a transcriptional repressor into an activator.

Desmoplastic Small Round Cell Tumors
Desmoplastic small round cell tumor (DSRCT) is a poorly understood malignant neoplasm with a predilection for adolescents and young adults that presents with widespread abdominal serosal involvement (27). This cancer is associated with a specific chromosomal abnormality, t(11;22)(p13;q12), involving the EWS and WT1 genes (8, 87, 91, 92).

The Wilms' tumor suppressor gene, WT1.
The WT1 tumor suppressor gene functions as a transcription factor and is implicated in the etiology of Wilms' tumor (WT), a pediatric malignancy of the kidney (16, 30). It encodes a zinc-finger protein, and sporadic mutations of this gene have been identified in about 10–15% of WT; germline mutations are associated with abnormal urogenital development and predisposition to WTs (73).

The WT1 gene spans ~50 kbp and contains 10 exons, the last four of which encode individual zinc fingers (Fig. 3A). The WT1 product is a ~50-kDa transcription factor with a proline- and glutamine-rich amino terminus and four zinc fingers of the Krüppel C2-H2 class at the carboxy terminus (16). The three carboxy-terminal-most zinc fingers share 67% identity with the three zinc fingers of early growth response gene-1 (EGR-1). The mRNA contains two alternatively spliced exons (31). The function of the first alternative splicing event (exon V) has not been well defined, although exon V can repress transcription when fused to a heterologous DNA-binding domain (105). Alternative splicing of exon IX inserts or removes three amino acids (±KTS) between zinc fingers III and IV and changes the DNA-binding specificity of WT1 (7, 82). The WT1(-KTS) isoforms can bind to two DNA motifs: 1) a GC-rich motif, 5'GG/YGTGGGCG/C3', similar to the EGR-1 binding site (82); and 2) a (5'TCC3')n-containing sequence (104). A role for WT1 in splicing has been postulated on the basis of the subnuclear localization of WT1(+KTS) and the interaction of this isoform with the U2AF65 splicing factor (22). A number of genes involved in growth regulation and cellular differentiation contain WT1 binding sites within their promoters, and their expression can be modulated by WT1 in transfection assays.



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Fig. 3. A: schematic diagram of the WT1 protein showing various functional domains. B: schematic diagram of the t(11;22)(p13;q12) of desmoplastic small round cell tumor (DSRCT) presenting the structure of normal EWS and WT1 genes, the EWS/WT1 fusion gene resulting from the chromosomal translocation, and the chimeric EWS/WT1 protein. Exon and intron structure of the EWS, WT1, and EWS/WT1 genes are shown with white or black boxes. As a result of alternative splicing, the EWS/WT1 chimeric gene encodes two different proteins. The alternative splicing event involves a splicing acceptor site in exon 9, causing the presence or absence of a 3-amino acid insertion (±KTS) between zinc fingers 3 and 4. Known functional domains of the EWS/WT1 oncoprotein are indicated.

 
The WT1 gene product has been shown to mediate both transcriptional repression and activation (83). Initial experiments focussing on transcriptional regulation by WT1 mediated via EGR-1 consensus sites demonstrated that WT1 is capable of repressing transcription; this effect is mediated by sequences within the NTD of WT1 (58). However, further characterization of the WT1 transcriptional properties and assessment of activity on putative cellular target genes revealed that WT1 can also activate transcription (103). Transcriptional regulation by WT1 appears complex, and a number of factors are known to influence the ability of WT1 to act as a repressor or activator (reviewed in Ref. 83). The transcriptional activity of WT1 can be modulated by interaction with two proteins: 1) the p53 tumor suppressor gene product (60) and 2) the Par-4 (prostate apoptosis response-4) protein (41), both interacting with the WT1 zinc fingers. Recently, physical association of WT1 with Hsp70 was documented, suggesting that Hsp70 may also play an important role in the regulation of WT1 (59). The WT1 isoforms can also self-associate (65, 84), and the major domain required for this maps to the first 182 amino acids of WT1 (Fig. 3A).

In addition, WT1 is a phosphorylated protein (90, 108). Ectopic expression of WT1 in fibroblasts results in in vivo phosphorylation of the protein, and treatment of cells with forskolin (an activator of cAMP-dependent protein kinase) induced phosphorylation of WT1. The forskolin-induced phosphorylation sites were identified as Ser-365 and Ser-393, amino acids that reside within the DNA-binding domain of zinc fingers 2 and 3, respectively (90). This phosphorylation abolishes the DNA-binding activity of WT1 in vitro (90) but leaves the RNA-binding activity unaltered (108).

EWS/WT1.
Molecular analysis of DSRCTs revealed that the product of the t(11;22)(p13;q12) translocation is a chimera containing the EWS NTD and three of the four WT1 CTD zinc fingers (Fig. 3B) (28, 53). The transcripts from the remaining wild-type EWS and WT1 genes are also produced in DSRCTs, as revealed by RT-PCR analysis (28). However, the product of the reciprocal translocation transcript, corresponding to the derivative chromosome 11 and consisting of the WT1 NTD and EWS CTD, is not detectable by RT-PCR (28).

The EWS/WT1 transcript retains the alternative splicing event between zinc fingers III and IV to produce two isoforms with fundamental differences in function (Fig. 3B) (28, 81). Introduction of EWS/WT1(-KTS) into NIH/3T3 cells results in increased growth rates, gain of anchorage-independent growth, and tumorigenicity in nude mice (46), whereas EWS/WT1(+KTS) had no phenotypic effects in these cells (46). Like its WT1 counterpart, the EWS/WT1(-KTS) isoform is able to form specific complexes with the WT1 recognition element (45, 81), whereas EWS/WT1(+KTS) cannot. Surprisingly, loss of zinc finger I in EWS/WT1(-KTS) results in a fusion product with higher DNA-binding affinity than the parental WT1(-KTS) product (45). WT1 zinc finger I appears to play a clear role in WT1 DNA recognition. Deletion of this finger, or site-directed mutagenesis affecting residues predicted to chelate zinc, decreases the efficiency of WT1/DNA complex formation (66). The importance of this finger to the function of WT1 is underscored by the identification of a germline missense mutation affecting a cysteine residue associated with Denys-Drash syndrome, a developmental disorder resulting in abnormal urogenital system development and predisposition to Wilms' tumors. Studies on the contribution to sequence recognition by Drummond et al. (25) suggest that zinc finger I contributes significantly to binding specificity.

It is thought that EWS/WT1(-KTS) might thus transform by binding to WT1 downstream targets and activating their transcription (46, 54). Candidate WT1 downstream targets (as determined by responsiveness of core promoter elements to exogenously supplied WT1 in transfection assays) include bcl-2, c-myc, EGFR (epidermal growth factor receptor), IGF-IR (insulin-like growth factor-I receptor), PDGF-A (platelet derived growth factor), IGF-II, CSF-1 (colony-stimulating factor), TGF ß1 (transforming growth factor), EGR-1 (early growth response-1), Pax-2, Pax-8, c-myb, G-protein {alpha}i-2, Ki-ras, insulin receptor, p21, Nov-H, RAR-{alpha}, Inhibin-{alpha}, syndecan-1, midkine, Dax-1, and WT1. The activity of some of these gene products are associated with oncogenic potential (e.g., autocrine and paracrine growth factors) and thus represent interesting targets for EWS/WT1(-KTS). Interestingly, PDGF-A has been documented to be overproduced in cell lines expressing EWS/WT1(-KTS), as well as in DSRCTs, and responds in vitro to EWS/WT1 (54). It is also possible that part of the transforming potential of EWS/WT1 is due to the ability of this fusion to bind to downstream targets of other zinc finger proteins that recognize motifs related to the WT1 binding site, such as members of the EGR-1 family.

As indicated above, the WT1 zinc fingers are not only required for nucleic acid recognition but also associate with a number of protein factors. We have noted that the Par-4 interaction is not maintained with either EWS/WT1 isoforms, raising the possibility that loss of this interaction may also be a contributing factor to the oncogenic potential of EWS/WT1 (46). The EWS NTD encodes a potent transcription activator, and deletion analysis has shown that the overall structure of the NTD contributes to the transactivation potential of EWS. Because Par-4 harbors a repression domain, it would be interesting to determine if reconstituting binding to Par-4 could prevent activation (and transformation) by EWS/WT1(-KTS).

Recently, we have found that EWS/WT1 is associated with the c-Abl protein tyrosine kinase in vivo (47). The c-abl gene product modifies EWS/WT1 by tyrosine phosphorylation, and this abrogates the DNA-binding ability of the fusion product (47). Regulation of the phosphorylation status of EWS/WT1 could provide a mechanism by which to abrogate the activity of this dominantly acting oncogene. In vitro and in vivo protein interaction experiments revealed that EWS/WT1 can also self-associate (47). The responsible domain maps to the fusion junction moiety of the chimeric protein and is negatively influenced by phosphorylation (47). Should self-association be essential to the transformation potential of EWS/WT1, then the responsible domain represents an ideal site against which to target an inhibitor, because the domain is a unique feature of the fusion product.

Malignant Melanoma of Soft Parts and t(12;22)(q13;q12)
Malignant melanoma of soft parts (MMSP) is a rare tumor that is typically associated with tendons and aponeuroses (tendinous expansion connecting a muscle with the parts that it moves) and is thought to be of neuroectodermal origin (17). In MMSP, a balanced t(12;22) translocation results in production of a fusion protein (EWS/ATF1) in which the amino-terminal 325 amino acids of EWS are fused to the carboxy-terminal 206 amino acids of activation transcription factor 1 (ATF1) (113). The reciprocal ATF1/EWS fusion protein is not produced in tumor cells because of an in-frame stop codon immediately carboxy-terminal to the ATF1 sequence (113).

ATF1 is a member of a group of bZIP transcription factors that includes the cAMP response element binding protein (CREB) and activator forms of the cAMP response element modulator (CREM) (55). ATF1 can homodimerize, heterodimerize with CREB, bind directly to cAMP-inducible promoters, and activate transcription upon phosphorylation by the cAMP-dependent protein kinase, PKA (86).

In contrast to ATF1, EWS/ATF1 functions as a potent constitutive activator of several cAMP-inducible promoters when assayed by transfection in cells lacking EWS/ATF1 (13, 26). In fact, EWS/ATF1 is a much more potent activator than EWS/Fli-1 compared with their corresponding nontumorigenic counterparts, ATF1 and Fli-1, respectively. EWS/ATF1 is ~200-fold more active than ATF1, whereas EWS/Fli-1 is only 5- to 10-fold more active than Fli-1 (13). Moreover, promoters that can be activated by exogeneous EWS/ATF1 are constitutively activated when introduced into tumor-derived cell lines that express EWS/ATF1 (13). These include promoters for somatostatin, c-fos, and vasoactive intestinal polypeptide (VIP). Thus EWS/ATF1 has the potential to upregulate cAMP-inducible promoters in MMSP cells, and this, in turn, might contribute to cellular transformation. To date, however, the activity of endogeneous cAMP-inducible promoters and the status of the cAMP-signaling pathway in MMSP cells has not been extensively studied.

Myxoid Liposarcoma and t(12;22)(q13;q12)
A few cases of myxoid liposarcomas, resulting in the hybrid gene TLS/FUS-CHOP, harbor a t(12;22)(q13;q12) rearrangement (71) (Table 1). This chromosomal translocation involves the CHOP gene (C/EBP homologous protein 10), a growth arrest and DNA-damage inducible member of the C/EBP family of transcription factors (89), residing on 12q13.1 (72). The CHOP gene (also referred to as GADD153) encodes a transcriptional regulator of the CCAAT/enhancer binding protein family (89). As a member of the leucine zipper transcription factor family, CHOP is implicated in adipocyte differentiation and growth arrest (15, 88). It is possible that the EWS/CHOP fusion product perturbs C/EBP function and causes a blockade in adipocyte differentiation.

Human Extraskeletal Myxoid Chondrosarcoma and t(9;22)(q22;q12)
The specific chromosomal translocation t(9;22)(q22;q12) has been observed in ~75% of the myxoid variant of chondrosarcoma (18, 52) (Table 1). An in-frame fusion of the 5' end of the EWS gene to a newly identified gene, TEC (translocated in extraskeletal myxoid chondrosarcoma, also known as CHN), on 9q22 was detected (18, 52). The fusion product consists of the EWS NTD linked to the entire TEC(CHN) coding region, consisting of a central DNA-binding domain and a carboxy-terminal ligand binding/dimerization domain (18). TEC(CHN) shows sequence homology to the Nur-related factor 1 family of orphan nuclear receptors that are involved in the control of cell proliferation and differentiation by modulating the response to growth factors and retinoic acid (34). The EWS/TEC(CHN) fusion protein is a potent transcriptional activator (51).

Sarcomas Harboring Translocations Involving EWS-Related Proteins
TLS/FUS and myxoid liposarcoma.
TLS (translocated in lymphosarcoma), also termed FUS (fusion), has extensive sequence similarity (55.6% identity) to the EWS gene (21). Like EWS, the NTD of TLS/FUS is rich in the amino acids glutamine, threonine, serine, proline, tyrosine, and glycine. It also contains multiple copies of the repeated hexapeptide (Ser/Gly)-Tyr-(Ser/Gly)-Gln-(Gln/Ser)-(Ser/Gln)-Pro. TLS/FUS is a potent transactivator when fused with various transcription factors (77). Both TLS/FUS and EWS contain a conserved 80-amino acid RNP core sequence within the CTD (43). Hence, TLS/FUS functions as an RNA-binding protein (21, 77) and binds specifically to poly(G) sequences (77). Both amino- and carboxy-terminal domains (that contain conserved RNA-binding motifs) of TLS/FUS are needed for poly(G)-specific RNA binding (77). TLS/FUS is bound to RNA in both the nucleus and the cytoplasm and is hypothesized to be a heterogeneous RNP-like chaperone for RNA (44, 112). TLS/FUS was found to interact with PU.1, an ETS protein capable of regulating transcription and RNA splicing. Overexpression of TLS/FUS in IW1–32 erythroid cells was shown to favor the use of a distal 5'-splicing site during E1A pre-mRNA splicing (32), suggesting that TLS/FUS may be part of a protein network involved in the regulation of RNA processing (32). Using the carboxy-terminal region of TLS/FUS as bait, two cDNAs encoding members of the serine-arginine (SR) family of proteins have been isolated (107). One cDNA corresponds to the mouse homolog of the human splicing factor SC35, and the second encodes a novel serine-arginine protein.

Like EWS, TLS/FUS is involved in various types of cancers through chromosomal translocation either with members of the ETS family or with other transcription (6). The TLS/FUS gene was originally identified in a human myxoid liposarcoma harboring a t(12;16) chromosomal translocation fusing it to the CHOP gene (21, 80). Approximately 75% of human myxoid liposarcomas contain a t(12;16)(q13;p11) chromosomal translocation (96, 101). This rearrangement fuses the CHOP gene to TLS/FUS (Table 1). In TLS/FUS-CHOP, the RNA-binding domain of TLS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP (21).

Consistent with the predicted oncogenic activity of the TLS/FUS-CHOP fusion gene, expression of TLS/FUS-CHOP in NIH/3T3 cells confers release from contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice (50, 111). Whereas the wild-type CHOP gene product interferes with the G1/S cell cycle progression, the TLS/FUS-CHOP gene product does not induce arrest at the G1/S checkpoint, but rather negatively interferes with the cell cycle regulatory activity of the wild-type CHOP gene product (3). Taken together, these observations suggest that the generation of TLS/FUS-CHOP fusion transcripts represents a critical event in the oncogenesis of an abundant subset of liposarcomas. These observations also indicate that the NTD of the EWS and TLS/FUS proteins are functionally equivalent when fused to CHOP because both products lead to the same tumor phenotype-myxoid liposarcoma (71) (Table 1).

TLS/FUS-ERG.
TLS/FUS also has been found fused to the ERG gene in human myeloid leukemias harboring a t(16;21)(p11;q22) chromosome translocation (37, 70, 93). Like EWS/ERG, TLS/FUS-ERG functions as a transcription activator (77), and the TLS/FUS fusion part contributes the activation domain to the TLS/FUS-ERG chimeric protein (77, 111). TLS/FUS-ERG shows weaker DNA-binding properties than the native ERG protein, and this translates into a weaker transactivation potential for the chimeric fusion (77). The contribution of the altered DNA-binding and transcriptional properties to the oncogenic potential of the TLS/FUS-chimeric proteins remains to be assessed. This fusion may sequester critical factors required for cell growth regulation through protein-protein interactions (26). In the same manner as with EWS/ERG, it has been shown that expression of TLS/FUS-ERG protein inhibits apoptosis of NIH/3T3 cells induced by either serum deprivation or by treatment with calcium ionophores. Inhibition of expression of TLS/FUS-ERG by antisense RNA resulted in increased susceptibility to apoptosis of tumor cells (109).

Other EWS-Related Proteins
hTAFII68.
The transcription factor hTAFII68 [human TBP (TATA-binding protein)-associated factorII68] shows extensive sequence similarity with both EWS and TLS/FUS (5). Like EWS and TLS/FUS, hTAFII68 contains a consensus RNA-binding domain that allows it to bind not only RNA but also single-stranded DNA. hTAFII68 was originally identified on the basis of its substoichiometric association with a distinct TFIID subpopulation. TFIID is a multiprotein complex composed of the TATA-binding protein (TBP) and TBP-associated factor (TAFIIs) and is the factor that nucleates preinitiation complex formation on protein-coding genes. Moreover, hTAFII68 can also associate with the RNA polymerase II complex. Interestingly, hTAFII68 is able to enter into the preinitiation complex together with RNA polymerase II, suggesting that hTAFII68 has a role in transcription initiation and/or elongation (5, 12, 39).

Interestingly, it has been shown that, TLS/FUS is associated with a subpopulation of TFIID complexes that are chromatographically distinct and functionally different from those containing hTAFII68 (5, 12, 39). These experiments strongly suggest that hTAFII68 and TLS/FUS may play an important role in the cross-talk between various components of the basal transcription machinery and that they may function by linking transcription initiation and elongation. Recently, it was also demonstrated that EWS is able to associate with the basal transcription factor TFIID (6). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Alternatively, the presence of EWS and hTAFII68 with the same RNA polymerase II complex may be complementary. Interestingly, EWS is not exclusively associated with TFIID but, similar to hTAFII68, is also associated with the RNA polymerase II complex (6).

SARFH/Cabeza.
A Drosophila protein, termed SARFH (sarcoma-associated RNA-binding fly homolog) or Cabeza, with a high degree of homology to EWS and TLS/FUS, has been described (38, 97). SARFH/Cabeza contains a conserved RNA-binding domain that shares homology with the same domain present in EWS, TLS/FUS, and hTAFII68, indicating that these belong to a new subfamily of RNP-containing proteins (5). SARFH/Cabeza was found to be associated with the majority of active transcription units in preparations of polytene chromosomes, indicating that, like other members of this family, it participates in the expression of genes transcribed by RNA polymerase II (38).


    CONCLUSIONS
 TOP
 ABSTRACT
 INTRODUCTION
 TRANSLOCATIONS INVOLVING THE EWS...
 CONCLUSIONS
 REFERENCES
 
The genetic changes that impart a growth advantage to the sarcomas described in this review have several common themes. 1) They involve the contribution of an NTD from a member of a novel class of nucleic acid-binding proteins that appear to be involved in transcription initiation and/or elongation. 2) A CTD containing a well-defined DNA-binding domain is contributed by a transcription factor. 3) The fusion product harbors unique properties that functionally distinguishes it from either parental gene from which it was derived. 4) Given that several types of sarcomas have specific translocations that result in the formation of chimeric genes encoding transcription factors, the specificity of transformation appears to be related to the DNA-binding domain acquired by the fusion product.

The identification of chromosomal breakpoints involving EWS or TLS/FUS genes has had a major impact on our understanding of gene products involved in transformation. The identification of target genes affected by these combinatorial proteins now represents the next challenge to help elucidate the tumorigenic pathways in which these proteins participate. The characterization of the chromosomal translocation breakpoints in sarcomas has also opened new avenues for molecular diagnostics. In addition, better functional characterization of the fusion proteins may lead to documentation of features unique to the chimeric product. Also, given the emerging picture that EWS and related proteins are present in different TFIID complexes, a better understanding into the normal biochemical activities of the EWS and TLS/FUS gene family would also provide important insight into the processes being usurped by the translocation products observed in these cancers.


    ACKNOWLEDGMENTS
 
Address for reprint requests and other correspondence: J. Pelletier, McIntyre Medical Science Building, McGill University, Rm 902, 3655 Drummond St., Montreal, Quebec, Canada H3G 1Y6 (E-mail: Jerry{at}med.mcgill.ca).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


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