A repetitive element containing a critical tyrosine residue is required for transcriptional activation by the EWS/ATF1 oncogene

Liang, Feng; Lee, Kevin A.W.

doi:10.1038/sj.onc.1204522

Cited by 25 publications

(46 citation statements)

References 43 publications

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“…We previously attempted to study the EAD by using various tricks. We were able to show that multiple cis-linked EAD sub-regions (~40 residues long) could create strong transcriptional activation domains [4] and that similarly small regions could synergise very effectively, in trans, on a promoter containing multiple activator binding sites [4]. The above findings provided evidence that the EAD harbors highly reiterated and flexible functional elements.…”

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confidence: 50%

Ewings family oncoproteins: drunk, disorderly and in search of partners

Lee

2007

Cell Res

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Cell Research | www.cell-research.com 286 npg COMMENTARY Aberrant chromosomal translocations involving the Ewings sarcoma (EWS) oncogene produce the EWS-fusionprotein family (EFPs, Figure 1A) that causes a wide variety of human cancers [1]. Extensive analysis of chromosomal breakpoints [2] has long established that, generally, EFPs contain at least the N-terminal 264 residues of EWS [the EWS-activation-domain (EAD)]. The C-terminal fusion partner is a cellular transcription factor that contributes, minimally, a sequence-specific DNA-binding domain which determines the tumor phenotype. Finally, EFPs are potent transcriptional activators but also interact with other proteins involved in different aspects of mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression.For many reasons the mechanism of EAD action has remained quite mysterious. One major barrier to progress is presented by the primary structure of the EAD ( Figure 1B) comprising ~250 residues and (almost exclusively) 30 copies of a degenerate hexapeptide repeat (DHR, consensus SYGQQS) with the Tyr being absolutely and the first Gln being strongly conserved ( Figure 1B). Because the intact EAD is required for full activity and considering its extended and repetitive sequence, it had not been feasible (until our study) to subject the EAD to systematic mutagenesis and to assess EAD structure/function relationships.The advance in technology (total gene synthesis) highlighted here [3] enabled us to overcome a long standing struggle but a brief comment on the history is fitting. We previously attempted to study the EAD by using various tricks. We were able to show that multiple cis-linked EAD sub-regions (~40 residues long) could create strong transcriptional activation domains [4] and that similarly small regions could synergise very effectively, in trans, on a promoter containing multiple activator binding sites [4]. The above findings provided evidence that the EAD harbors highly reiterated and flexible functional elements. However, whether the above properties of minimal synthetic activators could be extrapolated to the native EAD or to the biological functions of EFPs was a question that remained up in the air. Technology comes to the rescueThe advent of commercial gene synthesis enabled us to alter the entire EAD primary sequence at will and determine the functional consequences. We examined transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1 and made several notable findings in both cases [3]. First, multiple Tyr residues are specifically required. Second, an EAD in which every Tyr (a total of 38) was replaced by Phe retained function, establishing that an aromatic side chain is sufficient. Third, sequence inversions between adjacent Tyr residues also retained function indicating that overall sequence composition (and not the DHR) confers EAD activity. In summary, the particular amino acid composition of the EAD creates an enabling structure (due to its enrichment of Pro, Ser, Gln and Gly) with...

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confidence: 50%

Ewings family oncoproteins: drunk, disorderly and in search of partners

Lee

2007

Cell Res

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“…1B) suggests that DHRs, and particularly the conserved tyrosines therein, are critical for EAD function. A small region of the EAD (residues 8-41) has been directly characterized, and Tyr residues within this region are required for transactivation (12). Whether the above finding for transactivation can be extrapolated to the intact EAD and to EFP-induced oncogenic transformation has not been addressed.…”

Section: T He Ewings Sarcoma (Ews) Protooncogene Together Withmentioning

confidence: 99%

“…Tyr to Ile (DI, red boxes) and Tyr to Phe (DF and TF, blue boxes) are indicated. Protein 57Z contains EAD1-57 (including six DHRs) fused to ATF1 and the zta bZIP domain (12). 57ZA (A), 57ZI (I), 57ZF (F), 57ZW (W), and 57ZH (H) have all six DHR tyrosines changed to Ala, Ile, Phe, Trp, and His, respectively.…”

Section: Detailed Mutational Analysis Of the Eadmentioning

confidence: 99%

“…DI has reduced activity (Ϸ8% of WT) although residual activity is retained compared with DA (Ͻ1.6% of WT). Trp is the only remaining aromatic amino acid, but because of poor expression of Trp-enriched proteins, we used a different assay (12) in which a smaller protein (57Z) containing EAD1-57 (including six DHRs) fused to the zta bZIP domain efficiently can activate a multisite Zta-CAT reporter (12). 57Z is the control protein (Fig.…”

Section: Detailed Mutational Analysis Of the Eadmentioning

confidence: 99%

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Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins

Potikyan

Savene

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

152

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Chromosomal translocations involving the N-terminal Ϸ250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.Ewings sarcoma oncogene ͉ EWS/ATF1 ͉ intrinsically disordered proteins ͉ EWS activation domain ͉ molecular recognition feature T he Ewings sarcoma (EWS) protooncogene together with TLS/FUS and hTAF II 68 form a subgroup (the TET family; ref. 1) within the RNP family of RNA-binding proteins. Aberrant chromosomal translocations involving EWS produce EWS fusion proteins (EFPs) that cause human cancers (2, 3) and EFPs share the following common features (Fig. 1A). First, all EFPs contain at least the N-terminal 7 exons of EWS (residues 1-264) encoding the EWS activation domain (EAD). Second, the EWS fusion partner is one of several cellular transcription factors containing a sequence-specific DNA-binding domain that specifies the tumor type. Third, EFPs are potent transcriptional activators that depend on both the EAD and the fusion partner. Gene-specific transcriptional deregulation is likely to be a primary route for tumorigenesis (2, 3). However, some biological effects of EFPs might occur by alternative mechanisms including perturbation of pre-mRNA splicing (3).Progress in determining structure/function relations for the EAD has been limited for several reasons. The EAD has a highly repetitive primary sequence (Fig. 1B) containing multiple copies of a degenerate hexap...

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“…Activating transcription factor 1 is a bZIP protein that mediates cAMP-inducible transcription (Ribeiro et al, 1994), while in contrast EWS/ATF1 is a potent constitutive activator of cAMPinducible promoters (Brown et al, 1995;Fujimura et al, 1996;Pan et al, 1998;Feng and Lee, 2001); thus, suggesting that EWS/ATF1 may deregulate transcription of cAMP-inducible promoters in CCS cells. The physiological target promoters for EWS/ATF1 and their potential role in determining CCS biology and malignant transformation are beginning to be identified (Schaefer et al, 2002;Jishage et al, 2003).…”

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confidence: 99%

The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma

Goodall

Goding

et al. 2003

Br J Cancer

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Clear cell sarcoma (CCS) is associated with the EWS/ATF1 oncogene that is created by chromosomal fusion of the Ewings Sarcoma oncogene (EWS) and the cellular transcription factor ATF1. The melanocytic character of CCS suggests that the microphthalmiaassociated transcription factor (Mitf), a major inducer of melanocytic differentiation, may be miss-expressed in CCS. Accordingly, we show that the mRNA and protein of the melanocyte-specific isoform of Mitf (Mitf-M) are present in several cultured CCS cell lines (Su-ccs-1, DTC1, Kao, MST-1, MST-2 and MST-3). The above cell lines thus provide a valuable experimental resource for examining the role of Mitf-M in both CCS and melanocyte differentiation. Melanocyte-specific expression of Mitf-M is achieved via an ATFdependent melanocyte-specific cAMP-response element in the Mitf-M promoter, and expression of Mitf-M in CCS cells suggests that EWS/ATF1 (a potent and promiscuous activator of cAMP-inducible promoters) may activate the Mitf-M promoter. Surprisingly, however, the Mitf-M promoter is not activated by EWS/ATF1 in transient assays employing CCS cells, melanocytes or nonmelanocytic cells. Thus, our results indicate that Mitf-M promoter activation may require an appropriate chromosomal context in CCS cells or alternatively that the Mitf-M promoter is not directly activated by EWS/ATF1.

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A repetitive element containing a critical tyrosine residue is required for transcriptional activation by the EWS/ATF1 oncogene

Cited by 25 publications

References 43 publications

Ewings family oncoproteins: drunk, disorderly and in search of partners

Ewings family oncoproteins: drunk, disorderly and in search of partners

Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins

The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma

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