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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 PEA3-dependent target gene expression. (A) Silencing MED25 down-regulates the induction of MMP-1 mRNA by PEA3 in MCF-7 cells. RNA from cells transfected with control (Ctrl), control and MED25-specific siRNA (Ctrl + si MED25), PEA3 (PEA3) and PEA3 and MED25-specific siRNA (PEA3 + si MED25) was subjected to RT-qPCR. Relative expression levels were calculated using the ΔCt method using expression of GAPDH mRNA as a reference gene. Values are plotted relative to control set to 1. Data represent the mean ± S.E.M. of at least four independent transfections performed in duplicate. (Inset) The knockdown of endogenous MED25 and the expression of Flag-PEA3 were confirmed by western blot. (B) Effect of siRNA-mediated ETVs and MED25 depletion on MMP-1 expression in MDA-MB 231 cells. Data points are the average of three independent experiments. Error bars show standard deviation. (C) Effect of ETVs and MED25 depletion on Mediator occupancy at the MMP-1 promoter. ChIP experiments were performed using antibodies against MED1 and MED18 (ChIP MED) or IgG (ChIP Ctrl). ChIP/Input is average from two biological replicates. Error bars show standard deviation. Statistical significance was determined in paired Student’s t-tests (*P < 0.05).
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gkt199-F5: MED25 mediates PEA3-dependent target gene expression. (A) Silencing MED25 down-regulates the induction of MMP-1 mRNA by PEA3 in MCF-7 cells. RNA from cells transfected with control (Ctrl), control and MED25-specific siRNA (Ctrl + si MED25), PEA3 (PEA3) and PEA3 and MED25-specific siRNA (PEA3 + si MED25) was subjected to RT-qPCR. Relative expression levels were calculated using the ΔCt method using expression of GAPDH mRNA as a reference gene. Values are plotted relative to control set to 1. Data represent the mean ± S.E.M. of at least four independent transfections performed in duplicate. (Inset) The knockdown of endogenous MED25 and the expression of Flag-PEA3 were confirmed by western blot. (B) Effect of siRNA-mediated ETVs and MED25 depletion on MMP-1 expression in MDA-MB 231 cells. Data points are the average of three independent experiments. Error bars show standard deviation. (C) Effect of ETVs and MED25 depletion on Mediator occupancy at the MMP-1 promoter. ChIP experiments were performed using antibodies against MED1 and MED18 (ChIP MED) or IgG (ChIP Ctrl). ChIP/Input is average from two biological replicates. Error bars show standard deviation. Statistical significance was determined in paired Student’s t-tests (*P < 0.05).

Mentions: To evaluate the role of MED25 as a regulator of PEA3-dependent gene expression, we monitored the effect of MED25 depletion on PEA3-driven expression of two genes identified as targets of PEA3 members (24,37,38), namely MMP-1 and ICAM-1 (Figure 5). As a first functional approach, we used MCF-7 breast cancer cell line, which expresses low levels of PEA3 group members (21) as a model in which to overexpress PEA3. Expression of PEA3 was not affected by our MED25 siRNA, indicating the effect is specific to MED25 depletion (Figure 5A, Inset). Interestingly, silencing MED25 did not affect significantly ICAM-1 promoter activity or its activation by PEA3. However, it greatly diminished the transactivation of the MMP-1 promoter by PEA3. Importantly, similar results were obtained with ERM and ETV1 on MMP-1 and ICAM-1 activation (Supplementary Figure S4A), but in our hands, PEA3 was the most potent activator. This is in accordance with previous results (39).Figure 5.


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 PEA3-dependent target gene expression. (A) Silencing MED25 down-regulates the induction of MMP-1 mRNA by PEA3 in MCF-7 cells. RNA from cells transfected with control (Ctrl), control and MED25-specific siRNA (Ctrl + si MED25), PEA3 (PEA3) and PEA3 and MED25-specific siRNA (PEA3 + si MED25) was subjected to RT-qPCR. Relative expression levels were calculated using the ΔCt method using expression of GAPDH mRNA as a reference gene. Values are plotted relative to control set to 1. Data represent the mean ± S.E.M. of at least four independent transfections performed in duplicate. (Inset) The knockdown of endogenous MED25 and the expression of Flag-PEA3 were confirmed by western blot. (B) Effect of siRNA-mediated ETVs and MED25 depletion on MMP-1 expression in MDA-MB 231 cells. Data points are the average of three independent experiments. Error bars show standard deviation. (C) Effect of ETVs and MED25 depletion on Mediator occupancy at the MMP-1 promoter. ChIP experiments were performed using antibodies against MED1 and MED18 (ChIP MED) or IgG (ChIP Ctrl). ChIP/Input is average from two biological replicates. Error bars show standard deviation. Statistical significance was determined in paired Student’s t-tests (*P < 0.05).
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gkt199-F5: MED25 mediates PEA3-dependent target gene expression. (A) Silencing MED25 down-regulates the induction of MMP-1 mRNA by PEA3 in MCF-7 cells. RNA from cells transfected with control (Ctrl), control and MED25-specific siRNA (Ctrl + si MED25), PEA3 (PEA3) and PEA3 and MED25-specific siRNA (PEA3 + si MED25) was subjected to RT-qPCR. Relative expression levels were calculated using the ΔCt method using expression of GAPDH mRNA as a reference gene. Values are plotted relative to control set to 1. Data represent the mean ± S.E.M. of at least four independent transfections performed in duplicate. (Inset) The knockdown of endogenous MED25 and the expression of Flag-PEA3 were confirmed by western blot. (B) Effect of siRNA-mediated ETVs and MED25 depletion on MMP-1 expression in MDA-MB 231 cells. Data points are the average of three independent experiments. Error bars show standard deviation. (C) Effect of ETVs and MED25 depletion on Mediator occupancy at the MMP-1 promoter. ChIP experiments were performed using antibodies against MED1 and MED18 (ChIP MED) or IgG (ChIP Ctrl). ChIP/Input is average from two biological replicates. Error bars show standard deviation. Statistical significance was determined in paired Student’s t-tests (*P < 0.05).
Mentions: To evaluate the role of MED25 as a regulator of PEA3-dependent gene expression, we monitored the effect of MED25 depletion on PEA3-driven expression of two genes identified as targets of PEA3 members (24,37,38), namely MMP-1 and ICAM-1 (Figure 5). As a first functional approach, we used MCF-7 breast cancer cell line, which expresses low levels of PEA3 group members (21) as a model in which to overexpress PEA3. Expression of PEA3 was not affected by our MED25 siRNA, indicating the effect is specific to MED25 depletion (Figure 5A, Inset). Interestingly, silencing MED25 did not affect significantly ICAM-1 promoter activity or its activation by PEA3. However, it greatly diminished the transactivation of the MMP-1 promoter by PEA3. Importantly, similar results were obtained with ERM and ETV1 on MMP-1 and ICAM-1 activation (Supplementary Figure S4A), but in our hands, PEA3 was the most potent activator. This is in accordance with previous results (39).Figure 5.

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