A transcription assay for EWS oncoproteins in <italic>Xenopus</italic> oocytes

doi:10.1007/s13238-010-0114-y

Prot Cell

2010, Vol. 1

Issue (10) : 927-934 https://doi.org/10.1007/s13238-010-0114-y PMID: 21204019

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A transcription assay for EWS oncoproteins in Xenopus oocytes

King Pan Ng, Felix Cheung, Kevin A.W. Lee(

)

Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon Hong Kong, China

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Abstract

Aberrant chromosomal fusion of the Ewing's sarcoma oncogene (EWS) to several different cellular partners produces the Ewing's family of oncoproteins (EWS-fusion-proteins, EFPs) and associated tumors (EFTs). EFPs are potent transcriptional activators, dependent on the N-terminal region of EWS (the EWS-activation-domain, EAD) and this function is thought to be central to EFT oncogenesis and maintenance. Thus EFPs are promising therapeutic targets, but detailed molecular studies will be pivotal for exploring this potential. Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function. Therefore, the lack of manipulatable assays for the EAD presents a significant barrier to progress. Using Xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient, bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.

Keywords EWS/ATF1 Ewing's sarcoma microinjection Xenopus oocytes transcription EWS-activation domain

Corresponding Author(s): Lee Kevin A.W.,Email:bokaw@ust.hk

Issue Date: 01 October 2010

Cite this article:

King Pan Ng,Felix Cheung,Kevin A.W. Lee. A transcription assay for EWS oncoproteins in Xenopus oocytes[J]. Prot Cell, 2010, 1(10): 927-934.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-010-0114-y
https://academic.hep.com.cn/pac/EN/Y2010/V1/I10/927

Fig.1 Structures of EWS/ATF1 (EFPs), EAD, and experimental activators and reporters.
(A) Structure of EWS/ATF1 (EFPs). The Ewing's family of oncoproteins (EFPs) contains the N-terminal region of the Ewing's sarcoma oncogene (EWS) fused to various transcription factor partners (). All EFPs contain at least EWS residues 1-264 (the EWS-activation-domain, EAD) and a sequence-specific DNA binding domain from distinct partners. EWS/ATF1 is a typical EFP and contains EWS residues 1-325 fused to the C-terminal region of ATF1 (ΔATF1, residues 66-271), including the DNA binding (bZIP) domain (). EWS/ATF1 is a potent constitutive activator of ATF-dependent promoters () dependent on the EAD and the bZIP domain of ATF1. (B) Primary structure of the EAD. The EAD contains multiple degenerate hexapeptide repeats (DHRs, purple boxes) with consensus sequence SYGQQS. DHR degeneracy is indicated by the % occurrence of conserved residues including the absolutely conserved Tyr residue (red). Seven additional Tyr residues (dark gray boxes) are also present. Spaces between DHRs are generally only a few residues except in two cases (white boxes) of 12 and 25 residues, respectively. Several SH2 binding sites (YxxP, black circle) and SH3 binding sites (PxxP, open triangle) are indicated. (C) Structure of experimental activators and reporters. EZA contains the intact EAD (EWS residues 1-245), ΔATF1 and the zta bZIP domain (), ZΔE is equivalent to EZA but lacks the EAD. N3Z contains residues 1-166 of the EAD but is otherwise equivalent to EZA. Several Tyr residues within DHRs (purple boxes in N3Z) that are critical for transcriptional activation in mammalian cells () are shown. N3ZA and N3ZI are essentially the same as N3Z except that the Tyr residues highlighted (see materials and methods for precise coordinates) are changed to Ala (N3ZA, blue) or Isoleucine (N3ZI, orange). eN3Z corresponds to N3Z with EGFP fused to the N terminus and likewise for eN3ZA and eN3ZI (not shown). Expression vectors pSVEZA (), pZΔE (), pN3Z, pN3ZA and pN3ZI () were described elsewhere. All expression vectors were derived from pSG424 () containing the SV40 early promoter and polyadenylation signals for expression in oocytes. All proteins contained the KT3 epitope PPPEPET () at the C-terminus.

Fig.2 EAD-mediated trans-activation in oocytes.
(A) The germinal vesicle of unfertilised stage VI oocytes was injected with 2 ng of a Cat reporter plasmid (pZ7E4TCAT) containing seven zta binding sites either alone (control) or in the presence of 2 ng of vector (pSVEZA) expressing EZA (+EZA) containing the intact EAD. Cat assays were performed 40 h post-injection and an autoradiogram of the CAT assay is shown (c, chloramphenicol; ac, acetylated chloramphenicol). Each sample in the Cat assay shows the activity from three injected oocytes pooled together. (B) Oocytes were injected with 2 ng of a Luciferase reporter (pZ7Luc) containing seven zta binding sites and expression plasmids (2 ng) for EZA and Z?E or pGL3 (an SV40 promoter-Luciferase reporter). Luciferase reporter activity (kRLU/sec±SEM) was determined for individual oocytes and the mean values are shown (see Table 1 Exp#1) for uninjected oocytes (U), pZ7Luc alone, EZA, Z?E and pGL3. Western blot using KT3 antibody (left hand side) shows expression levels for EZA and ZΔE. Extract equivalent to 7 oocytes (derived from a total of 34 oocytes) was loaded on the gel.

Tab.1 Data summary

Fig.3 Analysis of activator levels and EAD activity in individual oocytes.
For pGL3 control reporter (blue bars, average activity 214±58 kRLU/sec) and activation of Z7Luc by eN3Z (green bars, average 78±17 kRLU/sec) the activity detected in ten individual oocytes is shown. The corresponding level of eN3Z expression for each oocyte (detected by Western blot using αEFGP JL8 antibody) is shown below the activity graph. Expression levels for eN3ZA and eN3ZI in individual oocytes (detected using αEFGP) is shown for the same experiment as eN3Z. eN3ZA and eN3ZI yield only background signals for trans-activation (Fig. 3C).

Fig.4 Analysis of N3Z and EGFP-N3Z proteins.
(A) Transcriptional activity of N3Z and N3ZA in mammalian cells was detected by Cat assay using the Z7Cat reporter. Western blot analysis using KT3 antibody shows activator expression levels. DNA binding activity was detected by gel mobility shift assay (right hand side). Extracts from transfected cells were incubated with 1ng of labeled probe containing a single zta binding site. DNA-protein complexes were resolved on non-denaturing polyacrylamide gels and detected by autoradiography. Positions of unbound DNA probe and DNA-protein complexes are indicated to the right. The presence of excess competitor oligonucleotide (100 ng) containing a functional (wt) or mutated (mt) zta binding site is indicated. (B) Transcriptional activity (kRLU/sec±SEM) of N3Z and derivatives in oocytes was determined as described in Fig. 2 using the Z7Luc reporter. The figure shows the individual data for each injected oocyte (left hand plot) and the mean values with S.E.M. (right hand side, see Table 1 Exp#2). (C) Transcriptional activity of EGFP-N3Z derivative (eN3Z) and mutants eN3ZA and eN3ZI was determined in mammalian cells by Cat assay using the Z7Cat reporter (left hand side). A Western blot using αEGFP antibody JL8 (Clontech) shows activator expression levels. The right hand graph shows the activity of eN3Z in oocytes using the Z7Luc reporter (mean values and SEM are shown, see Table 1 Exps#3-5). eN3ZA and eN3ZI both gave only background activity (b).

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