Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of MED25 on transcriptional activation by the ERM TAD. (A–D) U2OS cells were transfected with either Gal-ERM 1–72 (A, B and D) or full-length ERM (C) with (A and C) full length MED25, (B and C) MED25 ACID and MED25 VWA domains or (D) siRNA-resistant MED25 wild-type or derivatives. Cells collected 24 h after plasmid transfections were processed for luciferase activity. The relative luciferase activity of Gal-ERM 1–72 alone was assigned a transactivation level of 100%. Data represent the mean ± S.E.M. of at least three independent transfections performed in duplicate. (A) MED25 inhibits Gal4-ERM 1–72 transcriptional activation. Transient expression of MED25 in U2OS cells inhibits Gal4-ERM 1–72 transactivation but not activation by Gal4-E1A 13S or Gal4-E2F. (B) MED25 ACID and VWA domains inhibit Gal-ERM 1–72 transcriptional activation. (C) MED25 inhibits ERM transcriptional activity. (D) MED25 is required for full transcriptional activation by Gal4-ERM 1–72. siRNA-depleted cells were transfected with Gal-ERM 1–72 and reporters and analysed for luciferase activity. The expressed MED25 derivatives (WT, ΔACID, ΔCt, ΔNt, Q451E, M470A) were resistant to degradation induced by siRNA against wild-type MED25. The MED25 siRNA is directed against a sequence encoding the human ACID domain and therefore does not affect either the expression of MED25 ΔACID or the expression of MED25 WT, MED25 ΔCt, MED25 ΔNt, MED25 Q451E and MED25 M470A in which the human ACID domain has been replaced by its murine counterpart. (Inset) The levels of expression of MED25 after transient knockdown (as monitored by western blot with an antibody against MED25) and actin (loading control) are shown. The level of expression of the MED25 deletion mutants (western blot with anti-Flag antibody) after transfection into U2OS cells is also shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643604&req=5

gkt199-F4: Effects of MED25 on transcriptional activation by the ERM TAD. (A–D) U2OS cells were transfected with either Gal-ERM 1–72 (A, B and D) or full-length ERM (C) with (A and C) full length MED25, (B and C) MED25 ACID and MED25 VWA domains or (D) siRNA-resistant MED25 wild-type or derivatives. Cells collected 24 h after plasmid transfections were processed for luciferase activity. The relative luciferase activity of Gal-ERM 1–72 alone was assigned a transactivation level of 100%. Data represent the mean ± S.E.M. of at least three independent transfections performed in duplicate. (A) MED25 inhibits Gal4-ERM 1–72 transcriptional activation. Transient expression of MED25 in U2OS cells inhibits Gal4-ERM 1–72 transactivation but not activation by Gal4-E1A 13S or Gal4-E2F. (B) MED25 ACID and VWA domains inhibit Gal-ERM 1–72 transcriptional activation. (C) MED25 inhibits ERM transcriptional activity. (D) MED25 is required for full transcriptional activation by Gal4-ERM 1–72. siRNA-depleted cells were transfected with Gal-ERM 1–72 and reporters and analysed for luciferase activity. The expressed MED25 derivatives (WT, ΔACID, ΔCt, ΔNt, Q451E, M470A) were resistant to degradation induced by siRNA against wild-type MED25. The MED25 siRNA is directed against a sequence encoding the human ACID domain and therefore does not affect either the expression of MED25 ΔACID or the expression of MED25 WT, MED25 ΔCt, MED25 ΔNt, MED25 Q451E and MED25 M470A in which the human ACID domain has been replaced by its murine counterpart. (Inset) The levels of expression of MED25 after transient knockdown (as monitored by western blot with an antibody against MED25) and actin (loading control) are shown. The level of expression of the MED25 deletion mutants (western blot with anti-Flag antibody) after transfection into U2OS cells is also shown.

Mentions: To investigate whether the interaction between ERM and MED25 is functionally important, we first examined the influence of MED25 expression on the transactivation activity of ERM TAD 1–72 tethered to the Gal4 DNA-binding domain. It has been reported that overexpression of Mediator subunits can result in the inhibition of transcription owing to competition between the overexpressed subunits and the Mediator complex for the transcriptional activator (15,17,34,35). In agreement with these results, ectopic expression of MED25 efficiently inhibited the transcriptional activity of ERM TAD (Figure 4A). By contrast, overexpression of MED25 had no influence on the transcriptional activity of the transcription factor E2F or the Adenovirus E1A protein (Figure 4A), the established target of which in Mediator is MED23 (34). Thus, the influence of MED25 in this context appears to be specific to ERM. Similar interference was observed with two MED25 truncated fragments VWA and ACID (Figure 4B), suggesting that the Mediator-binding domain (VWA) and the ERM-binding domain (ACID) of MED25 are functionally important for mediating the transcriptional activity of ERM. Interestingly, this behaviour of ERM is reminiscent of the reported effects of MED25 on VP16 transcriptional activity (14). We also sought to test if full-length ERM activity is affected by MED25 and chose the well-known Ets-responsive TORU-luciferase reporter construct (36). We found that full-length MED25 as well as MED25-VWA and MED25-ACID domains significantly reduced ERM transcriptional activity (Figure 4C). These results were in total agreement with those obtained with the Gal4 fusions used above.Figure 4.


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)

Effects of MED25 on transcriptional activation by the ERM TAD. (A–D) U2OS cells were transfected with either Gal-ERM 1–72 (A, B and D) or full-length ERM (C) with (A and C) full length MED25, (B and C) MED25 ACID and MED25 VWA domains or (D) siRNA-resistant MED25 wild-type or derivatives. Cells collected 24 h after plasmid transfections were processed for luciferase activity. The relative luciferase activity of Gal-ERM 1–72 alone was assigned a transactivation level of 100%. Data represent the mean ± S.E.M. of at least three independent transfections performed in duplicate. (A) MED25 inhibits Gal4-ERM 1–72 transcriptional activation. Transient expression of MED25 in U2OS cells inhibits Gal4-ERM 1–72 transactivation but not activation by Gal4-E1A 13S or Gal4-E2F. (B) MED25 ACID and VWA domains inhibit Gal-ERM 1–72 transcriptional activation. (C) MED25 inhibits ERM transcriptional activity. (D) MED25 is required for full transcriptional activation by Gal4-ERM 1–72. siRNA-depleted cells were transfected with Gal-ERM 1–72 and reporters and analysed for luciferase activity. The expressed MED25 derivatives (WT, ΔACID, ΔCt, ΔNt, Q451E, M470A) were resistant to degradation induced by siRNA against wild-type MED25. The MED25 siRNA is directed against a sequence encoding the human ACID domain and therefore does not affect either the expression of MED25 ΔACID or the expression of MED25 WT, MED25 ΔCt, MED25 ΔNt, MED25 Q451E and MED25 M470A in which the human ACID domain has been replaced by its murine counterpart. (Inset) The levels of expression of MED25 after transient knockdown (as monitored by western blot with an antibody against MED25) and actin (loading control) are shown. The level of expression of the MED25 deletion mutants (western blot with anti-Flag antibody) after transfection into U2OS cells is also shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643604&req=5

gkt199-F4: Effects of MED25 on transcriptional activation by the ERM TAD. (A–D) U2OS cells were transfected with either Gal-ERM 1–72 (A, B and D) or full-length ERM (C) with (A and C) full length MED25, (B and C) MED25 ACID and MED25 VWA domains or (D) siRNA-resistant MED25 wild-type or derivatives. Cells collected 24 h after plasmid transfections were processed for luciferase activity. The relative luciferase activity of Gal-ERM 1–72 alone was assigned a transactivation level of 100%. Data represent the mean ± S.E.M. of at least three independent transfections performed in duplicate. (A) MED25 inhibits Gal4-ERM 1–72 transcriptional activation. Transient expression of MED25 in U2OS cells inhibits Gal4-ERM 1–72 transactivation but not activation by Gal4-E1A 13S or Gal4-E2F. (B) MED25 ACID and VWA domains inhibit Gal-ERM 1–72 transcriptional activation. (C) MED25 inhibits ERM transcriptional activity. (D) MED25 is required for full transcriptional activation by Gal4-ERM 1–72. siRNA-depleted cells were transfected with Gal-ERM 1–72 and reporters and analysed for luciferase activity. The expressed MED25 derivatives (WT, ΔACID, ΔCt, ΔNt, Q451E, M470A) were resistant to degradation induced by siRNA against wild-type MED25. The MED25 siRNA is directed against a sequence encoding the human ACID domain and therefore does not affect either the expression of MED25 ΔACID or the expression of MED25 WT, MED25 ΔCt, MED25 ΔNt, MED25 Q451E and MED25 M470A in which the human ACID domain has been replaced by its murine counterpart. (Inset) The levels of expression of MED25 after transient knockdown (as monitored by western blot with an antibody against MED25) and actin (loading control) are shown. The level of expression of the MED25 deletion mutants (western blot with anti-Flag antibody) after transfection into U2OS cells is also shown.
Mentions: To investigate whether the interaction between ERM and MED25 is functionally important, we first examined the influence of MED25 expression on the transactivation activity of ERM TAD 1–72 tethered to the Gal4 DNA-binding domain. It has been reported that overexpression of Mediator subunits can result in the inhibition of transcription owing to competition between the overexpressed subunits and the Mediator complex for the transcriptional activator (15,17,34,35). In agreement with these results, ectopic expression of MED25 efficiently inhibited the transcriptional activity of ERM TAD (Figure 4A). By contrast, overexpression of MED25 had no influence on the transcriptional activity of the transcription factor E2F or the Adenovirus E1A protein (Figure 4A), the established target of which in Mediator is MED23 (34). Thus, the influence of MED25 in this context appears to be specific to ERM. Similar interference was observed with two MED25 truncated fragments VWA and ACID (Figure 4B), suggesting that the Mediator-binding domain (VWA) and the ERM-binding domain (ACID) of MED25 are functionally important for mediating the transcriptional activity of ERM. Interestingly, this behaviour of ERM is reminiscent of the reported effects of MED25 on VP16 transcriptional activity (14). We also sought to test if full-length ERM activity is affected by MED25 and chose the well-known Ets-responsive TORU-luciferase reporter construct (36). We found that full-length MED25 as well as MED25-VWA and MED25-ACID domains significantly reduced ERM transcriptional activity (Figure 4C). These results were in total agreement with those obtained with the Gal4 fusions used above.Figure 4.

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