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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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MED25 mediates the interaction between ERM and Mediator. (A) Depletion of endogenous MED25 from DAMI cell nuclear extract using Halo-Tag fusion protein ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L. After incubation and extensive washing, MED25 not associated with ERM 1–72 present in the supernatant was saved as the unbound fraction (U). Samples were separated by SDS-PAGE, and MED25 was detected by immunoblotting with anti-MED25 antibody. I: Input, 10% of the nuclear extracts used in binding reactions. U: Unbound, 10%. (B) MED25 and Mediator (here documented with MED1 and MED6) bind specifically to ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L as in Figure 3A. ERM 1–72-bound Mediator complex (B) was eluted by boiling the beads in SDS buffer and was resolved by 12% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10% of the nuclear extracts used in binding reactions. B: bound material, 100%. (C) MED25 depletion of nuclear extract impairs recruitment of Mediator to ERM. Nuclear extracts from DAMI cells were sequentially incubated with Halo-Tag ERM 1–72 as indicated. After incubation and extensive washing, the supernatant (containing unbound material, U1, U2 and U3) and the affinity resin (containing bound material, B1, B2 and B3) were boiled in SDS buffer and resolved by SDS-PAGE. Specifically bound and unbound proteins were detected by western blot using the specified antibodies. I: input, 10%. U: unbound, 10%. B: bound, 100%. (D) MED25 immunodepletion reduces interaction between ERM and Mediator. Anti-MED25 antibody and non-specific anti-serum (control) were first coupled to Pureproteome protein A magnetic beads (Millipore). Nuclear extracts from DAMI cells were then incubated at 4°C with antibody-bound protein A beads. The resulting supernatant was incubated with immobilized Halo-Tag ERM 1–72 as indicated. ID: immunodepletion. Bound (B) and unbound (U) materials were resolved by 10% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10%. U: unbound, 10% and B: bound, 100%.
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gkt199-F3: MED25 mediates the interaction between ERM and Mediator. (A) Depletion of endogenous MED25 from DAMI cell nuclear extract using Halo-Tag fusion protein ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L. After incubation and extensive washing, MED25 not associated with ERM 1–72 present in the supernatant was saved as the unbound fraction (U). Samples were separated by SDS-PAGE, and MED25 was detected by immunoblotting with anti-MED25 antibody. I: Input, 10% of the nuclear extracts used in binding reactions. U: Unbound, 10%. (B) MED25 and Mediator (here documented with MED1 and MED6) bind specifically to ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L as in Figure 3A. ERM 1–72-bound Mediator complex (B) was eluted by boiling the beads in SDS buffer and was resolved by 12% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10% of the nuclear extracts used in binding reactions. B: bound material, 100%. (C) MED25 depletion of nuclear extract impairs recruitment of Mediator to ERM. Nuclear extracts from DAMI cells were sequentially incubated with Halo-Tag ERM 1–72 as indicated. After incubation and extensive washing, the supernatant (containing unbound material, U1, U2 and U3) and the affinity resin (containing bound material, B1, B2 and B3) were boiled in SDS buffer and resolved by SDS-PAGE. Specifically bound and unbound proteins were detected by western blot using the specified antibodies. I: input, 10%. U: unbound, 10%. B: bound, 100%. (D) MED25 immunodepletion reduces interaction between ERM and Mediator. Anti-MED25 antibody and non-specific anti-serum (control) were first coupled to Pureproteome protein A magnetic beads (Millipore). Nuclear extracts from DAMI cells were then incubated at 4°C with antibody-bound protein A beads. The resulting supernatant was incubated with immobilized Halo-Tag ERM 1–72 as indicated. ID: immunodepletion. Bound (B) and unbound (U) materials were resolved by 10% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10%. U: unbound, 10% and B: bound, 100%.

Mentions: To determine whether the ERM TAD interacts with intact Mediator, we incubated nuclear extracts of DAMI cells with purified Halo-Tag ERM 1–72 coupled to magnetic beads. Unbound Mediator subunits present in the supernatant after incubation (U for unbound) and beads-bound Mediator subunits (B for bound) were then monitored by immunoblotting with antibodies to the subunits indicated in the figure (Figure 3). Firstly, endogenous MED25 could be almost completely depleted from the nuclear extracts after incubation with Halo-Tag ERM 1–72 but not with Halo-Tag alone, confirming that ERM interacts very efficiently with MED25 (Figure 3A). Notably this depletion was not seen when we introduced a mutation in ERM (ERM 1–72 F47L) that prevents MED25 binding (Figure 2B). Accordingly, endogenous MED25 was only detected in the fraction bound to wild-type ERM 1–72 (Figure 3B). Secondly, we observed that MED1 and MED6 bound Halo-Tag ERM 1–72 but not Halo-Tag alone, thus revealing a specific association of ERM with Mediator (Figure 3B). Importantly, as expected, incubation with the mutant ERM 1–72 F47L impaired the interaction between Mediator and ERM (Figure 3B). Finally, when the nuclear extracts were sequentially incubated with ERM 1–72, we observed a strong correlation between depletion of MED25 and decrease in MED1, MED6, MED14 and MED24 binding (Figure 3C, compare B1, B2 and B3).Figure 3.


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)

MED25 mediates the interaction between ERM and Mediator. (A) Depletion of endogenous MED25 from DAMI cell nuclear extract using Halo-Tag fusion protein ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L. After incubation and extensive washing, MED25 not associated with ERM 1–72 present in the supernatant was saved as the unbound fraction (U). Samples were separated by SDS-PAGE, and MED25 was detected by immunoblotting with anti-MED25 antibody. I: Input, 10% of the nuclear extracts used in binding reactions. U: Unbound, 10%. (B) MED25 and Mediator (here documented with MED1 and MED6) bind specifically to ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L as in Figure 3A. ERM 1–72-bound Mediator complex (B) was eluted by boiling the beads in SDS buffer and was resolved by 12% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10% of the nuclear extracts used in binding reactions. B: bound material, 100%. (C) MED25 depletion of nuclear extract impairs recruitment of Mediator to ERM. Nuclear extracts from DAMI cells were sequentially incubated with Halo-Tag ERM 1–72 as indicated. After incubation and extensive washing, the supernatant (containing unbound material, U1, U2 and U3) and the affinity resin (containing bound material, B1, B2 and B3) were boiled in SDS buffer and resolved by SDS-PAGE. Specifically bound and unbound proteins were detected by western blot using the specified antibodies. I: input, 10%. U: unbound, 10%. B: bound, 100%. (D) MED25 immunodepletion reduces interaction between ERM and Mediator. Anti-MED25 antibody and non-specific anti-serum (control) were first coupled to Pureproteome protein A magnetic beads (Millipore). Nuclear extracts from DAMI cells were then incubated at 4°C with antibody-bound protein A beads. The resulting supernatant was incubated with immobilized Halo-Tag ERM 1–72 as indicated. ID: immunodepletion. Bound (B) and unbound (U) materials were resolved by 10% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10%. U: unbound, 10% and B: bound, 100%.
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gkt199-F3: MED25 mediates the interaction between ERM and Mediator. (A) Depletion of endogenous MED25 from DAMI cell nuclear extract using Halo-Tag fusion protein ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L. After incubation and extensive washing, MED25 not associated with ERM 1–72 present in the supernatant was saved as the unbound fraction (U). Samples were separated by SDS-PAGE, and MED25 was detected by immunoblotting with anti-MED25 antibody. I: Input, 10% of the nuclear extracts used in binding reactions. U: Unbound, 10%. (B) MED25 and Mediator (here documented with MED1 and MED6) bind specifically to ERM 1–72. Nuclear extracts from DAMI cells were incubated with immobilized Halo-Tag, Halo-Tag ERM 1–72 and Halo-Tag ERM 1–72 F47L as in Figure 3A. ERM 1–72-bound Mediator complex (B) was eluted by boiling the beads in SDS buffer and was resolved by 12% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10% of the nuclear extracts used in binding reactions. B: bound material, 100%. (C) MED25 depletion of nuclear extract impairs recruitment of Mediator to ERM. Nuclear extracts from DAMI cells were sequentially incubated with Halo-Tag ERM 1–72 as indicated. After incubation and extensive washing, the supernatant (containing unbound material, U1, U2 and U3) and the affinity resin (containing bound material, B1, B2 and B3) were boiled in SDS buffer and resolved by SDS-PAGE. Specifically bound and unbound proteins were detected by western blot using the specified antibodies. I: input, 10%. U: unbound, 10%. B: bound, 100%. (D) MED25 immunodepletion reduces interaction between ERM and Mediator. Anti-MED25 antibody and non-specific anti-serum (control) were first coupled to Pureproteome protein A magnetic beads (Millipore). Nuclear extracts from DAMI cells were then incubated at 4°C with antibody-bound protein A beads. The resulting supernatant was incubated with immobilized Halo-Tag ERM 1–72 as indicated. ID: immunodepletion. Bound (B) and unbound (U) materials were resolved by 10% SDS-PAGE before western blot analysis using the specified antibodies. I: input, 10%. U: unbound, 10% and B: bound, 100%.
Mentions: To determine whether the ERM TAD interacts with intact Mediator, we incubated nuclear extracts of DAMI cells with purified Halo-Tag ERM 1–72 coupled to magnetic beads. Unbound Mediator subunits present in the supernatant after incubation (U for unbound) and beads-bound Mediator subunits (B for bound) were then monitored by immunoblotting with antibodies to the subunits indicated in the figure (Figure 3). Firstly, endogenous MED25 could be almost completely depleted from the nuclear extracts after incubation with Halo-Tag ERM 1–72 but not with Halo-Tag alone, confirming that ERM interacts very efficiently with MED25 (Figure 3A). Notably this depletion was not seen when we introduced a mutation in ERM (ERM 1–72 F47L) that prevents MED25 binding (Figure 2B). Accordingly, endogenous MED25 was only detected in the fraction bound to wild-type ERM 1–72 (Figure 3B). Secondly, we observed that MED1 and MED6 bound Halo-Tag ERM 1–72 but not Halo-Tag alone, thus revealing a specific association of ERM with Mediator (Figure 3B). Importantly, as expected, incubation with the mutant ERM 1–72 F47L impaired the interaction between Mediator and ERM (Figure 3B). Finally, when the nuclear extracts were sequentially incubated with ERM 1–72, we observed a strong correlation between depletion of MED25 and decrease in MED1, MED6, MED14 and MED24 binding (Figure 3C, compare B1, B2 and B3).Figure 3.

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