RGG-boxes of the EWS oncoprotein repress a range of transcriptional activation domains

Abstract

The Ewings Sarcoma Oncoprotein (EWS) interacts with several components of the mammalian transcriptional and pre-mRNA splicing machinery and is also found in the cytoplasm and even on the cell surface. The apparently diverse cellular functions of EWS are, however, not well characterized. EWS harbours a potent N-terminal transcriptional activation domain (the EAD) that is revealed in the context of oncogenic EWS-fusion proteins (EFPs) and a C-terminal RNA-binding domain (RBD) that recruits pre-mRNA splicing factors and may couple transcription and splicing. In contrast to EFPs, the presumed transcriptional role of normal EWS remains enigmatic. Here, we report that multiple RGG-boxes within the RBD are necessary and sufficient for cis-repression of the EAD and that RGG-boxes can also repress in-trans, within dimeric partners. Lys can functionally substitute for Arg, indicating that the basic nature of the Arg side chain is the critical determinant of RGG-box-mediated repression. In addition to the EAD, RGG-boxes can repress a broad range of activation domains (including those of VP16, E1a and CREB), but repression can be alleviated by the simultaneous presence of more than one activation domain. We therefore propose that a key function of RGG boxes within native EWS is to restrict promiscuous activation by the EAD while still allowing EWS to enter functional transcription complexes and participate in other transactions involving pre-mRNAs.

Figure 1

Functional regions of EWS, ATF1 and EWS/ATF1. EWS contains an N-terminal transcriptional activation domain (the EWS-Activation Domain, EAD) and a C-terminal RNA-binding domain (RBD). The RBD contains two elements [an RNA-Recognition Motif (RRM) and RGG boxes] commonly found in RNA binding proteins (–8) and a C2–C2 putative zinc finger (Z). The RRM (otherwise called the CS-RBD or the RNP motif) consists of ∼100 residues with a conserved three dimensional structure (–8). The three RGG rich regions (RGG1, RGG2 and RGG3) contain 5, 3 and 12 tripeptide RGG motifs, respectively. The term RGG-box refers to clusters of closely spaced RGG tripeptides flanked by aromatic residues and a consensus RGG-box (containing 5 RGG tri-peptides) has been deduced (8). ATF1 is a PKA-inducible activator (47,48). The bZIP domain of ATF1 (amino acids 214–271) mediates dimerization and DNA-binding, Q2 is a glutamine-rich constitutive activation domain (48) and PKA represents the Kinase Inducible Domain (48) including a single PKA-phosphoacceptor site. EWS/ATF1 is an oncogenic EWS-Fusion-Protein (EFP) that is associated with soft tissue clear cell sarcoma (CCS) (4). The chromosomal cross-over point that produces EWS/ATF1 is shown with a black cross resulting in the EAD (residues 1–325) fused to the C-terminal region of ATF1 (residues 66–271). EWS/ATF1 lacks the PKA phosphoacceptor site of ATF1 and functions as a potent constitutive activator of ATF-dependent promoters (20,21) dependent on the EAD and the bZIP domain of ATF1.

Figure 2

An assay for repression by small regions of the EWS RBD. (A) Structure of experimental proteins. For reference, the structure of EWS, ATF1 and EWS/ATF1 are described in Figure 1. Δ87C (45) is a derivative of the EWS/ATF1 oncogene containing only the N-terminal 86 residues of the EAD fused to ATF1 (residues 66–271) including the bZIP domain that allows dimerization and DNA binding. Δ87C is a constitutive activator of ATF-dependent promoters dependent on the EAD region (45). RGG3 corresponds to residues 545–656 of intact EWS. All of the novel proteins tested are derived from Δ87C by insertion of the indicated sequence between the EAD and ATF1 regions. The ATF1 region is thus present in all proteins but is not shown in the diagram. The amino acid sequence of a short repeat (SR, light green box) and a long repeat (LR, dark green box) are present within the RGG3 region of EWS (LR contains EWS residues 571–604 and SR contains EWS residues 587–604). The number of RGG-tripeptides in each protein and the relative transcription activity are shown to the right. (B) Transcription assays. JEG3 cells were transfected with 5 μg of ATF-dependent reporter [pΔ−(71)SomCat] and 5 μg of plasmid expressing the activator indicated. Transcriptional activity was monitored by CAT assay and a representative TLC result is shown at the bottom (c = chloramphenicol; and ac = acetylated chloramphenicol). The corresponding western blot, using monoclonal antibody KT3 (45) to monitor epitope-tagged activator levels, is shown above the CAT assay.

Figure 3

Mutational analysis of SR4. Residues present in each mutant are aligned with the SR4 amino acid sequence. RGG tri-peptides are highlighted by green boxes and point mutations are in red. The relative transcription activity for each mutant (compared with Δ87C set at 100) is indicated to the right. For experiments, JEG3 cells were transfected with 5 μg of ATF-dependent reporter [pΔ−(71)SomCat] and 5 μg of plasmid expressing the indicated activator. Transcriptional activity was monitored by CAT assay. Middle panel. western blot analysis of KT3 epitope-tagged proteins in transfected cells. Lower panel: representative TLC of CAT assay (c = chloramphenicol; and ac = acetylated chloramphenicol).

Figure 4

Specificity and dominance of RGG-box mediated repression. (A) Effect of the RBD on single activation domains (EAD, VP16, E1a and CREB). (B) Effect of RBD on two activation domains (EAD and VP16) present together. JEG3 cells were transfected with plasmids expressing the proteins indicated (as described in Materials and Methods) together with the following reporter constructs: p(Δ− 71)SomCat (for activators containing the ATF1 DNA-binding domain); pBS2CAT (for activators containing the BSAP DNA-binding domain); pG5E4TCAT (for G4E1a and E1aR) and pG1E4TCAT (for VP16 and derivatives). CAT assays were performed 40 h post-transfection and a representative CAT assay is shown. Relative transcription activity is shown in parenthesis to the right of each protein tested. For single activation domains (A) activity in the absence of the RBD is set at 100. For the joint activity of VP16 and the EAD (B) the activity of G4VP16 is set at 100.

Figure 5

Effect of the RBD in obligatory heterodimers. JEG3 cells were transfected with a Zta reporter plasmid (pZ7E4TCAT) together with two other plasmids expressing either a bZ12 derivative (as indicated to the left) or bZ3 derivative (indicated to the right). Thus, the resultant dimeric proteins in each case will consist of a heterodimer containing one bZ12 partner and one bZ3 partner. CAT assays were performed 40 h post-transfection and a representative CAT assay is shown. Relative transcription activity for each heterodimer is shown in the right hand column with the bZ3EA/bZ12EA combination set at 100. nd, not determined.