Acetylation Increases EWS-FLI1 DNA Binding and Transcriptional Activity

Abstract

Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

Keywords: EWS-FLI1; Ewing’s sarcoma; PCAF; acetylation.

Figure 1

Acetylases increase transcriptional and DNA binding activity of EWS-FLI1. (A) The EWS-FLI1 responsive NR0B1 promoter (Gangwal et al., 2008) and pCI-EWS-FLI1 were co-transfected into Cos7 cells without PCAF nor p300 (white bar) or with increasing amounts of either PCAF or p300 (gray bars) or a mutant HAT construct lacking the histone acetyltransferase domain (PCAF ΔHAT, black bar). Twenty-four hours later promoter derived luciferase activity as well as protein levels were analyzed. Bar graphs result from triplicate transfection and duplicate luciferase measurement. EWS-FLI1 levels did not exhibit any patterns of change across multiple experiments and those changes here were not representative and thus not considered in final conclusions. (B) In vitro acetylation was carried out as described and samples of the reaction were tested for successful acetylation by western blot using an anti-acetyl-lysine antibody. (C) Recombinant, refolded FLI1-CTD specifically binds to the ets-binding site of dsDNA. The specificity of the FLI1-CTD for the ets-binding site in the oligonucleotide was proven by the lack of binding to mutant oligonucleotide (lane 8), by the competition of unlabeled wild type oligonucleotide (lanes 2–4), and by the lack of competition with mutant oligonucleotide (lanes 5–7); comp, competitor; bio, biotinylated. (D) Samples were subjected to non-radioactive EMSA using biotin labeled ets binding site as a probe. Unlabeled wild type competitor was used at 1, 2.5, 5, 10, and 50 times excess. Mutant competitor was only used at the highest concentration (50 times excess). (E) Aliquots of the reactions were tested for equal loading by Coomassie staining. (F) Densitometric analysis of (B,C). Relative binding = EMSA signal/Coomassie signal.

Figure 2

Full-length EWS-FLI1, and its C-terminal FLI1 domain (FLI1-CTD) become acetylated in vitro by p300/CBP and PCAF. Recombinant, refolded full-length FLI1 (ERG B), FLI-CTD, EWS-FLI1, and EWS were subjected to in vitro acetylation (C¹⁴-AcetylCoA), PAGE, and autoradiography using p300 (A), PCAF (B), and CBP (C). Autoacetylation of the acetyltransferases serves as internal positive control. (D) Densitometric analysis of the band intensities shown in A–C using the densitometric values/area (Q/pixel2). These acetylations were replications were: the full length EWS and FLI1, EWS-FLI1 each performed twice, the FLI-CTD greater than 10 replicates.

Figure 3

Identification of individual acetylation sites in FLI1-CTD by mass spectrometry. (A) Sequence of the C-terminal FLI1 domain (FLI1-CTD) as present in all EWS-FLI1 fusion types. Top numbers (BOLD) correspond to amino acid positions in the wild type FLI1 protein; lower numbers refer to positions in the EWS-FLI1 (type I) fusion protein. Lysine residues identified as being acetylated are marked in red and their numbering corresponds to the wild type FLI1 sequence. The ets-domain is boxed in yellow. (B) Lysine positions in the full-length wild type FLI1 and the corresponding lysine positions in the three different EWS-FLI1 fusion proteins. (C) Results of MS analysis of in vitro acetylated FLI1-CTD protein. Recombinant, refolded FLI-CTD was subjected to in vitro acetylation by CBP and p300 (no enzyme as control). Trypsin generated peptides were analyzed using ESI LC MS/MS (QStarELITE/TEMPO MDLC system) MS as described in the Section “Experimental Procedures.” The lysine positions most abundantly found as being acetylated are listed.

Figure 4

Mutagenesis confirms K240, K252, and K380 as major acetylation sites. (A) Two lysine to arginine mutants were created of the FLI1-CTD of EWS-FLI1 using site directed mutagenesis. K4bR (K240,252,380,397R) and K9aR (K325,327,334,345,350,354,359,380, 397R). (B) Protein sequence alignment of different ets family members at relevant lysine positions. Protein sequences of ETS1 (P14921), ETS2 (P15036), ETV6 (P41212), ETV7 (Q9Y603), SPIB (Q01892), SPI1 (P17947), ETV1 (P50549), ETV5 (P41161), ETV4 (P43268), ERG (P11308), FLI1 (Q01543), FEV (Q99581), and ETV2 (O00321) were aligned using UniProt, to reveal conservation of Lysine positions K240, K252, K380, and K397. (C,D) Acetylation mutants were in vitro acetylated by CBP (C) or PCAF (D). (E) Kinetic of CBP in vitro acetylation of K4bR and K380, 397R double mutant proteins. (F) Densitometric analysis of (E) (wt, filled squares; K4bR, open triangles; K380,397R double mutant, open circles). For in vitro acetylations dried gels were re-hydrated after exposure and stained for Coomassie to show equal loading [lower panels (B–D,F)]. This experiment was performed only once in this kinetic fashion. (G) Non-radioactive EMSA using ets-binding site containing oligonucleotides and mutant recombinant proteins. Fractions of the EMSA samples were analyzed by Coomassie (lower panel). (H) Densitometric analysis of images shown in (G). Relative binding = EMSA signal/Coomassie signal.

Figure 5

Histone acetyltransferases are expressed in ES cells and directly interact with EWS-FLI1. (A) Whole cell lysates of various ES cells were tested for expression of CBP, p300, and PCAF protein by western blot. (B) EWS-FLI1 was immunoprecipitated from A4573 cells with a FLI1 antibody to detect acetylation by western blot using a pan-α-acetyl lysine. (C) Recombinant EWS-FLI1 directly binds to recombinant p300 (upper panel) and recombinant PCAF (lower panel). One microgram of each recombinant protein was used. (D) TC32 cells were transfected with PCAF expression plasmid. After 24 h lysates were subjected to immunoprecipitation with FLI1 antibody to show that the EWS-FLI1 PCAF complex also occurs in ES cells. (E) TC32 cells were transfected with PCAF followed by treatment with 0.3 μM TSA for 16 h. Whole cell lysates were immunoprecipitated with an anti-FLI1 antibody. A pool of site-specific anti-acetyl-EWS-FLI1 antibodies was used to detect acetyl-EWS-FLI1 (antibodies to K240Ac, K252Ac, K380Ac, K397Ac. For antibody generation, refer to see Experimental Procedures). The same membrane was stripped and reprobed with α-FLI1 and α-actin.

Figure 6

Specificity of EWS-FLI1 acetylation site-specific antibodies. FLI1-CTD protein was in vitro acetylated using non-radioactive acetyl-CoA and CBP (+) or non-radioactive acetyl-CoA without CBP (−). Aliquots of the same samples were separated on SDS PAGE and strips were incubated with antibodies as indicated. For peptide competition antibodies were incubated with 1 μg/ml of K240Ac, K252Ac, and K380Ac or 5 μg/ml K395Ac peptides.

Figure 7

EWS-FLI1 acetylation increases upon HDI treatment and co-expression of PCAF. (A) Cos 7 cells were co-transfected with EWS-FLI1 type I and PCAF-expression plasmid or empty vectors to show that EWS-FLI1 interacts with PCAF in Cos 7 cells. Thirty hours post transfection cells were lysed and lysate was subjected to immunoprecipitation with α-FLI1 or IgG antibodies. Western blots were probed with α-FLI1 and α-PCAF antibodies. (B) Cos 7 cells were transfected with wt EWS-FLI1, with or without PCAF. Acetylated EWS-FLI1 was detected by single site-specific antibodies K240Ac and K380Ac. (C) Cos 7 cells were transfected with wt EWS-FLI1 or EF-K4bR constructs, with or without PCAF. (D) Cos 7 cells were transfected using pCIneo empty vector, wt-EWS-FLI1, or its acetylation mutant EF-K4bR in combination with pcDNA-4TO empty vector or pcDNA4TO-PCAF and the NR0B1 reporter construct. The next day cells were treated with SAHA (2 μM) or TSA (0.3 μM) for 8 h (control: untreated) and then subjected to luciferase and western blot analysis. Luciferase activity was expressed as luciferase activity (sample) – luciferase activity (empty vector control) containing both empty vectors (pcDNA4TO and pciNeo). Bar graphs result from triplicate transfection and duplicate luciferase measurement. Two-tailed t-test was performed using GraphPad Prism version 4.00.