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The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members.

Verger A, Baert JL, Verreman K, Dewitte F, Ferreira E, Lens Z, de Launoit Y, Villeret V, Monté D - Nucleic Acids Res. (2013)

Bottom Line: Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM.Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment.In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.

View Article: PubMed Central - PubMed

Affiliation: IRI USR 3078 CNRS, Parc CNRS de la Haute Borne, 50 avenue de Halley, B.P. 70478, 59658 Villeneuve d'Ascq Cedex, France.

ABSTRACT
PEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro. Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.

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Effect of mutations in the ERM/MED25 interface. (A) Mutation of Q451 of MED25 severely reduces its ability to bind to ERM. (Left) GST ERM 38–72 was used to analyse the binding to full-length Flag-MED25 WT and point mutants Q451E and M470A. Q451E shows weak binding to ERM 38–72. Binding was detected by immunoblotting with anti-Flag. An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (Right) Wild-type Flag-MED25 or MED25 with a Q451E mutation were examined for their ability to interact with ERM in co-immunoprecipitation experiments. RK13 cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag. Expression of Flag-MED25, Flag-MED25 Q451E and ERM are shown in the top two panels. (B) Mutation of F47 of ERM abolishes the recruitment of MED25. (Left) GST MED25 ACID domain was examined for its ability to interact in vitro with ERM wild type or ERM F47L mutant. (Right) The ability of Flag-MED25 to interact with ERM wild type and with ERM F47L mutant was analysed in co-immunoprecipitation experiments. Cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag or IB α ERM (IB). Expression of Flag-MED25, ERM and ERM F47L are shown in the top panel (cellular extract).
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gkt199-F2: Effect of mutations in the ERM/MED25 interface. (A) Mutation of Q451 of MED25 severely reduces its ability to bind to ERM. (Left) GST ERM 38–72 was used to analyse the binding to full-length Flag-MED25 WT and point mutants Q451E and M470A. Q451E shows weak binding to ERM 38–72. Binding was detected by immunoblotting with anti-Flag. An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (Right) Wild-type Flag-MED25 or MED25 with a Q451E mutation were examined for their ability to interact with ERM in co-immunoprecipitation experiments. RK13 cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag. Expression of Flag-MED25, Flag-MED25 Q451E and ERM are shown in the top two panels. (B) Mutation of F47 of ERM abolishes the recruitment of MED25. (Left) GST MED25 ACID domain was examined for its ability to interact in vitro with ERM wild type or ERM F47L mutant. (Right) The ability of Flag-MED25 to interact with ERM wild type and with ERM F47L mutant was analysed in co-immunoprecipitation experiments. Cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag or IB α ERM (IB). Expression of Flag-MED25, ERM and ERM F47L are shown in the top panel (cellular extract).

Mentions: Because the residues of ERM 38–72 that form the MED25 interface display sequence homology with the VP16 H1 subdomain (Supplementary Figure S1A), we anticipated that mutations affecting the MED25/ERM and MED25/VP16 complexes would exhibit similar behaviours and indeed, found this to be the case. First, a MED25 point mutant (Q451E) that has been shown previously to abolish MED25/VP16 interaction (31) also greatly compromised the ability of MED25 to interact with ERM TAD in GST pull-down assay and in co-immunoprecipitation experiments (Figure 2A). Conversely, an M470A mutation that had no effect on ERM binding (Figure 2A), similarly failed to impair VP16 binding (data not shown). In addition, the MED25 ACID domain by itself or full-length MED25 failed to bind a mutated ERM activation domain where the F47 residue was changed to leucine (Figure 2B). It is notable that the mutation of this residue strongly impaired ERM transactivation (3) (Supplementary Figure S1B). Remarkably, the corresponding residue in VP16 H1 (F442, see Supplementary Figure S1A) has previously been shown to be critical for the transactivation function of VP16 (10) and for the recruitment of MED25 (14,15,33). Our results thus demonstrate that MED25 binds only to a functional ERM TAD.Figure 2.


The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members.

Verger A, Baert JL, Verreman K, Dewitte F, Ferreira E, Lens Z, de Launoit Y, Villeret V, Monté D - Nucleic Acids Res. (2013)

Effect of mutations in the ERM/MED25 interface. (A) Mutation of Q451 of MED25 severely reduces its ability to bind to ERM. (Left) GST ERM 38–72 was used to analyse the binding to full-length Flag-MED25 WT and point mutants Q451E and M470A. Q451E shows weak binding to ERM 38–72. Binding was detected by immunoblotting with anti-Flag. An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (Right) Wild-type Flag-MED25 or MED25 with a Q451E mutation were examined for their ability to interact with ERM in co-immunoprecipitation experiments. RK13 cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag. Expression of Flag-MED25, Flag-MED25 Q451E and ERM are shown in the top two panels. (B) Mutation of F47 of ERM abolishes the recruitment of MED25. (Left) GST MED25 ACID domain was examined for its ability to interact in vitro with ERM wild type or ERM F47L mutant. (Right) The ability of Flag-MED25 to interact with ERM wild type and with ERM F47L mutant was analysed in co-immunoprecipitation experiments. Cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag or IB α ERM (IB). Expression of Flag-MED25, ERM and ERM F47L are shown in the top panel (cellular extract).
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gkt199-F2: Effect of mutations in the ERM/MED25 interface. (A) Mutation of Q451 of MED25 severely reduces its ability to bind to ERM. (Left) GST ERM 38–72 was used to analyse the binding to full-length Flag-MED25 WT and point mutants Q451E and M470A. Q451E shows weak binding to ERM 38–72. Binding was detected by immunoblotting with anti-Flag. An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (Right) Wild-type Flag-MED25 or MED25 with a Q451E mutation were examined for their ability to interact with ERM in co-immunoprecipitation experiments. RK13 cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag. Expression of Flag-MED25, Flag-MED25 Q451E and ERM are shown in the top two panels. (B) Mutation of F47 of ERM abolishes the recruitment of MED25. (Left) GST MED25 ACID domain was examined for its ability to interact in vitro with ERM wild type or ERM F47L mutant. (Right) The ability of Flag-MED25 to interact with ERM wild type and with ERM F47L mutant was analysed in co-immunoprecipitation experiments. Cells were transfected with the indicated expression vectors and cellular extracts were IP α Flag and IB α Flag or IB α ERM (IB). Expression of Flag-MED25, ERM and ERM F47L are shown in the top panel (cellular extract).
Mentions: Because the residues of ERM 38–72 that form the MED25 interface display sequence homology with the VP16 H1 subdomain (Supplementary Figure S1A), we anticipated that mutations affecting the MED25/ERM and MED25/VP16 complexes would exhibit similar behaviours and indeed, found this to be the case. First, a MED25 point mutant (Q451E) that has been shown previously to abolish MED25/VP16 interaction (31) also greatly compromised the ability of MED25 to interact with ERM TAD in GST pull-down assay and in co-immunoprecipitation experiments (Figure 2A). Conversely, an M470A mutation that had no effect on ERM binding (Figure 2A), similarly failed to impair VP16 binding (data not shown). In addition, the MED25 ACID domain by itself or full-length MED25 failed to bind a mutated ERM activation domain where the F47 residue was changed to leucine (Figure 2B). It is notable that the mutation of this residue strongly impaired ERM transactivation (3) (Supplementary Figure S1B). Remarkably, the corresponding residue in VP16 H1 (F442, see Supplementary Figure S1A) has previously been shown to be critical for the transactivation function of VP16 (10) and for the recruitment of MED25 (14,15,33). Our results thus demonstrate that MED25 binds only to a functional ERM TAD.Figure 2.

Bottom Line: Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM.Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment.In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.

View Article: PubMed Central - PubMed

Affiliation: IRI USR 3078 CNRS, Parc CNRS de la Haute Borne, 50 avenue de Halley, B.P. 70478, 59658 Villeneuve d'Ascq Cedex, France.

ABSTRACT
PEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro. Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.

Show MeSH
Related in: MedlinePlus