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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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ERM binds to MED25. (A) Schematic summary of the interaction between ERM and MED25 proteins. The N-terminal TAD of ERM interacts with the ACID of MED25. NR: Nuclear receptor box. Numbers refer to amino acid. (B) Deletion analysis of ERM shows that the N-terminal 38–72 domain is sufficient for binding MED25 in vitro. GST fusion proteins of the indicated ERM fragments were used to assess the binding to full-length rabbit reticulocyte lysate in vitro generated Flag-MED25. Binding was detected by autoradiography (upper panel) or immunoblotting with anti-Flag (bottom panel). An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (C) Deletion analysis of MED25 shows that the ACID domain is sufficient for binding to ERM 38–72 in vitro. GST and GST-ERM 38–72 were incubated with the indicated MED25 fragments produced in reticulocyte lysate. Binding was detected by immunoblotting with anti-Flag or anti-GFP. (D) Co-immunoprecipitation of MED25 with ERM. Flag-MED25 and wild-type or mutants ERM (upper panel) or mutants Flag-MED25 together with full-length ERM (bottom panel) were expressed in RK13 cells. Cellular extracts were immunoprecipitated with anti-Flag antibody (IP α Flag) and immunoblotted with anti-Flag (IB α Flag) and anti-ERM (IB α ERM) antibodies. Aliquots of the same extracts were analysed with the same antibodies to detect exogenous proteins (cellular extract). (E) Interaction between endogenous proteins. MDA-MB 231 nuclear extracts were subjected to immunoprecipitation with anti-Gal4 DBD (IP Ctrl) or anti-ERM (IP ERM) antibodies. Interactions were detected by western blot using polyclonal anti-MED25 (IB α MED25) or polyclonal anti-ERM (IB α ERM).
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gkt199-F1: ERM binds to MED25. (A) Schematic summary of the interaction between ERM and MED25 proteins. The N-terminal TAD of ERM interacts with the ACID of MED25. NR: Nuclear receptor box. Numbers refer to amino acid. (B) Deletion analysis of ERM shows that the N-terminal 38–72 domain is sufficient for binding MED25 in vitro. GST fusion proteins of the indicated ERM fragments were used to assess the binding to full-length rabbit reticulocyte lysate in vitro generated Flag-MED25. Binding was detected by autoradiography (upper panel) or immunoblotting with anti-Flag (bottom panel). An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (C) Deletion analysis of MED25 shows that the ACID domain is sufficient for binding to ERM 38–72 in vitro. GST and GST-ERM 38–72 were incubated with the indicated MED25 fragments produced in reticulocyte lysate. Binding was detected by immunoblotting with anti-Flag or anti-GFP. (D) Co-immunoprecipitation of MED25 with ERM. Flag-MED25 and wild-type or mutants ERM (upper panel) or mutants Flag-MED25 together with full-length ERM (bottom panel) were expressed in RK13 cells. Cellular extracts were immunoprecipitated with anti-Flag antibody (IP α Flag) and immunoblotted with anti-Flag (IB α Flag) and anti-ERM (IB α ERM) antibodies. Aliquots of the same extracts were analysed with the same antibodies to detect exogenous proteins (cellular extract). (E) Interaction between endogenous proteins. MDA-MB 231 nuclear extracts were subjected to immunoprecipitation with anti-Gal4 DBD (IP Ctrl) or anti-ERM (IP ERM) antibodies. Interactions were detected by western blot using polyclonal anti-MED25 (IB α MED25) or polyclonal anti-ERM (IB α ERM).

Mentions: The human ERM protein belongs to the PEA3 subfamily of Ets proteins (2) and contains at least four functional domains (Figure 1A): an amino-terminal transactivation domain (TAD; residues 1–72) (3,4), a central negative regulatory domain (NRD; residues 73–298) (5,6), the carboxy-terminal ETS domain (residues 363–451) and a carboxy-terminal TAD (residues 452–510) (4). Initial experiments demonstrated that the first 72 residues and the carboxy-terminal tail constituted transferrable activation domains (4). Subsequent experiments demonstrated that the amino-terminal TAD is regulated by a flanking NRD, which functions in a sumoylation-dependent manner (5,6). The amino-terminal activation domain is highly conserved (∼85% sequence identity) among PEA3 subfamily members and represents the main activation domain of all three proteins (3,4,7,8). The TAD from the protein ERM displays minimal stable tertiary structure (9), and the combination of acidic and hydrophobic amino acids within this domain appears similar to that found in the TADs of other activators such as the herpes simplex viral protein 16 (VP16) (10,11).Figure 1.


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)

ERM binds to MED25. (A) Schematic summary of the interaction between ERM and MED25 proteins. The N-terminal TAD of ERM interacts with the ACID of MED25. NR: Nuclear receptor box. Numbers refer to amino acid. (B) Deletion analysis of ERM shows that the N-terminal 38–72 domain is sufficient for binding MED25 in vitro. GST fusion proteins of the indicated ERM fragments were used to assess the binding to full-length rabbit reticulocyte lysate in vitro generated Flag-MED25. Binding was detected by autoradiography (upper panel) or immunoblotting with anti-Flag (bottom panel). An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (C) Deletion analysis of MED25 shows that the ACID domain is sufficient for binding to ERM 38–72 in vitro. GST and GST-ERM 38–72 were incubated with the indicated MED25 fragments produced in reticulocyte lysate. Binding was detected by immunoblotting with anti-Flag or anti-GFP. (D) Co-immunoprecipitation of MED25 with ERM. Flag-MED25 and wild-type or mutants ERM (upper panel) or mutants Flag-MED25 together with full-length ERM (bottom panel) were expressed in RK13 cells. Cellular extracts were immunoprecipitated with anti-Flag antibody (IP α Flag) and immunoblotted with anti-Flag (IB α Flag) and anti-ERM (IB α ERM) antibodies. Aliquots of the same extracts were analysed with the same antibodies to detect exogenous proteins (cellular extract). (E) Interaction between endogenous proteins. MDA-MB 231 nuclear extracts were subjected to immunoprecipitation with anti-Gal4 DBD (IP Ctrl) or anti-ERM (IP ERM) antibodies. Interactions were detected by western blot using polyclonal anti-MED25 (IB α MED25) or polyclonal anti-ERM (IB α ERM).
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gkt199-F1: ERM binds to MED25. (A) Schematic summary of the interaction between ERM and MED25 proteins. The N-terminal TAD of ERM interacts with the ACID of MED25. NR: Nuclear receptor box. Numbers refer to amino acid. (B) Deletion analysis of ERM shows that the N-terminal 38–72 domain is sufficient for binding MED25 in vitro. GST fusion proteins of the indicated ERM fragments were used to assess the binding to full-length rabbit reticulocyte lysate in vitro generated Flag-MED25. Binding was detected by autoradiography (upper panel) or immunoblotting with anti-Flag (bottom panel). An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (C) Deletion analysis of MED25 shows that the ACID domain is sufficient for binding to ERM 38–72 in vitro. GST and GST-ERM 38–72 were incubated with the indicated MED25 fragments produced in reticulocyte lysate. Binding was detected by immunoblotting with anti-Flag or anti-GFP. (D) Co-immunoprecipitation of MED25 with ERM. Flag-MED25 and wild-type or mutants ERM (upper panel) or mutants Flag-MED25 together with full-length ERM (bottom panel) were expressed in RK13 cells. Cellular extracts were immunoprecipitated with anti-Flag antibody (IP α Flag) and immunoblotted with anti-Flag (IB α Flag) and anti-ERM (IB α ERM) antibodies. Aliquots of the same extracts were analysed with the same antibodies to detect exogenous proteins (cellular extract). (E) Interaction between endogenous proteins. MDA-MB 231 nuclear extracts were subjected to immunoprecipitation with anti-Gal4 DBD (IP Ctrl) or anti-ERM (IP ERM) antibodies. Interactions were detected by western blot using polyclonal anti-MED25 (IB α MED25) or polyclonal anti-ERM (IB α ERM).
Mentions: The human ERM protein belongs to the PEA3 subfamily of Ets proteins (2) and contains at least four functional domains (Figure 1A): an amino-terminal transactivation domain (TAD; residues 1–72) (3,4), a central negative regulatory domain (NRD; residues 73–298) (5,6), the carboxy-terminal ETS domain (residues 363–451) and a carboxy-terminal TAD (residues 452–510) (4). Initial experiments demonstrated that the first 72 residues and the carboxy-terminal tail constituted transferrable activation domains (4). Subsequent experiments demonstrated that the amino-terminal TAD is regulated by a flanking NRD, which functions in a sumoylation-dependent manner (5,6). The amino-terminal activation domain is highly conserved (∼85% sequence identity) among PEA3 subfamily members and represents the main activation domain of all three proteins (3,4,7,8). The TAD from the protein ERM displays minimal stable tertiary structure (9), and the combination of acidic and hydrophobic amino acids within this domain appears similar to that found in the TADs of other activators such as the herpes simplex viral protein 16 (VP16) (10,11).Figure 1.

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