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Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

Hamard PJ, Boyer-Guittaut M, Camuzeaux B, Dujardin D, Hauss C, Oelgeschläger T, Vigneron M, Kedinger C, Chatton B - Nucleic Acids Res. (2007)

Bottom Line: This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID.In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo.Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institut Gilbert Laustriat, Ecole Supérieure de Biotechnologie de Strasbourg, UMR7175 CNRS-ULP, BP10413, 67412 Strasbourg Illkirch Cedex, France.

ABSTRACT
Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

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Sumoylation affects ATF7 transcriptional activity. (A) HeLa-SUMO cells were co-transfected with 0.25 µg of the pG4-ATF7 expression vectors, 2 µg of the luciferase reporter, 0.25 µg of the TBP-spm3 and 0.25 µg of p-hsTAF12 expression vectors. The structures of the pG4-ATF7 activator and the luciferase reporter plasmids are schematized. The results of luciferase assays are presented (arbitrary units). (B) HeLa-SUMO cells were transfected with 0.5 µg px-ATF7 derivatives and hsTAF12. Extracts from transfected cells were immunoprecipitated (IP) with the anti-ATF7 (2F10) monoclonal antibody as described in Materials and Methods. The purified complexes were separated by SDS-PAGE. The presence of hsTAF12 proteins in both IP and extracts, and ATF7 derivatives in the extracts was revealed by western blotting (WB) using specific antibodies.
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Figure 5: Sumoylation affects ATF7 transcriptional activity. (A) HeLa-SUMO cells were co-transfected with 0.25 µg of the pG4-ATF7 expression vectors, 2 µg of the luciferase reporter, 0.25 µg of the TBP-spm3 and 0.25 µg of p-hsTAF12 expression vectors. The structures of the pG4-ATF7 activator and the luciferase reporter plasmids are schematized. The results of luciferase assays are presented (arbitrary units). (B) HeLa-SUMO cells were transfected with 0.5 µg px-ATF7 derivatives and hsTAF12. Extracts from transfected cells were immunoprecipitated (IP) with the anti-ATF7 (2F10) monoclonal antibody as described in Materials and Methods. The purified complexes were separated by SDS-PAGE. The presence of hsTAF12 proteins in both IP and extracts, and ATF7 derivatives in the extracts was revealed by western blotting (WB) using specific antibodies.

Mentions: The (17m5)-TK-Luc reporter (19) contains the luciferase gene, driven by the thymidine kinase promoter and five Gal4 binding sites (see Figure 5A).


Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

Hamard PJ, Boyer-Guittaut M, Camuzeaux B, Dujardin D, Hauss C, Oelgeschläger T, Vigneron M, Kedinger C, Chatton B - Nucleic Acids Res. (2007)

Sumoylation affects ATF7 transcriptional activity. (A) HeLa-SUMO cells were co-transfected with 0.25 µg of the pG4-ATF7 expression vectors, 2 µg of the luciferase reporter, 0.25 µg of the TBP-spm3 and 0.25 µg of p-hsTAF12 expression vectors. The structures of the pG4-ATF7 activator and the luciferase reporter plasmids are schematized. The results of luciferase assays are presented (arbitrary units). (B) HeLa-SUMO cells were transfected with 0.5 µg px-ATF7 derivatives and hsTAF12. Extracts from transfected cells were immunoprecipitated (IP) with the anti-ATF7 (2F10) monoclonal antibody as described in Materials and Methods. The purified complexes were separated by SDS-PAGE. The presence of hsTAF12 proteins in both IP and extracts, and ATF7 derivatives in the extracts was revealed by western blotting (WB) using specific antibodies.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851647&req=5

Figure 5: Sumoylation affects ATF7 transcriptional activity. (A) HeLa-SUMO cells were co-transfected with 0.25 µg of the pG4-ATF7 expression vectors, 2 µg of the luciferase reporter, 0.25 µg of the TBP-spm3 and 0.25 µg of p-hsTAF12 expression vectors. The structures of the pG4-ATF7 activator and the luciferase reporter plasmids are schematized. The results of luciferase assays are presented (arbitrary units). (B) HeLa-SUMO cells were transfected with 0.5 µg px-ATF7 derivatives and hsTAF12. Extracts from transfected cells were immunoprecipitated (IP) with the anti-ATF7 (2F10) monoclonal antibody as described in Materials and Methods. The purified complexes were separated by SDS-PAGE. The presence of hsTAF12 proteins in both IP and extracts, and ATF7 derivatives in the extracts was revealed by western blotting (WB) using specific antibodies.
Mentions: The (17m5)-TK-Luc reporter (19) contains the luciferase gene, driven by the thymidine kinase promoter and five Gal4 binding sites (see Figure 5A).

Bottom Line: This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID.In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo.Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institut Gilbert Laustriat, Ecole Supérieure de Biotechnologie de Strasbourg, UMR7175 CNRS-ULP, BP10413, 67412 Strasbourg Illkirch Cedex, France.

ABSTRACT
Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

Show MeSH
Related in: MedlinePlus