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Identification of the gene transcription repressor domain of Gli3.

Tsanev R, Tiigimägi P, Michelson P, Metsis M, Østerlund T, Kogerman P - FEBS Lett. (2008)

Bottom Line: We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally.Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3.This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia.

ABSTRACT
Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

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HDAC recruitment study of the Gli3 repressor domain. HEK293 cells were transfected with Gli3-RD (squares) or the repressor domain of REST (positive control, triangles) and treated with increasing amounts of the HDAC inhibitor TSA. As negative control we used cells transfected with empty vector (diamonds). The analyzes were performed three to six times and the error bars indicate standard deviations.
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fig4: HDAC recruitment study of the Gli3 repressor domain. HEK293 cells were transfected with Gli3-RD (squares) or the repressor domain of REST (positive control, triangles) and treated with increasing amounts of the HDAC inhibitor TSA. As negative control we used cells transfected with empty vector (diamonds). The analyzes were performed three to six times and the error bars indicate standard deviations.

Mentions: The suggested Ski binding site on Gli3 has not been exactly mapped (it is in the region from residue 152 to 397 [9]) and may overlap with the identified repressor part. Since Ski and SUFU recruits HDACs to exert their inhibitory role, we wanted to test if the repressor function described here depends on the same mechanism or not. We transfected HEK293 cells with either Gli3-RD or the repressor domain of REST (that serve as positive control since it depend on HDAC to repress transcription) and tested the effect of the HDAC inhibitor TSA as shown in Fig. 4. Again the Gli3-RD suppresses transcription but there is not any effect of TSA up to 1 μM. In contrast the repression by REST is relieved at only 200 nM TSA. This shows that Gli3-RD induced repression is not dependent on HDACs in the way REST is.


Identification of the gene transcription repressor domain of Gli3.

Tsanev R, Tiigimägi P, Michelson P, Metsis M, Østerlund T, Kogerman P - FEBS Lett. (2008)

HDAC recruitment study of the Gli3 repressor domain. HEK293 cells were transfected with Gli3-RD (squares) or the repressor domain of REST (positive control, triangles) and treated with increasing amounts of the HDAC inhibitor TSA. As negative control we used cells transfected with empty vector (diamonds). The analyzes were performed three to six times and the error bars indicate standard deviations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697317&req=5

fig4: HDAC recruitment study of the Gli3 repressor domain. HEK293 cells were transfected with Gli3-RD (squares) or the repressor domain of REST (positive control, triangles) and treated with increasing amounts of the HDAC inhibitor TSA. As negative control we used cells transfected with empty vector (diamonds). The analyzes were performed three to six times and the error bars indicate standard deviations.
Mentions: The suggested Ski binding site on Gli3 has not been exactly mapped (it is in the region from residue 152 to 397 [9]) and may overlap with the identified repressor part. Since Ski and SUFU recruits HDACs to exert their inhibitory role, we wanted to test if the repressor function described here depends on the same mechanism or not. We transfected HEK293 cells with either Gli3-RD or the repressor domain of REST (that serve as positive control since it depend on HDAC to repress transcription) and tested the effect of the HDAC inhibitor TSA as shown in Fig. 4. Again the Gli3-RD suppresses transcription but there is not any effect of TSA up to 1 μM. In contrast the repression by REST is relieved at only 200 nM TSA. This shows that Gli3-RD induced repression is not dependent on HDACs in the way REST is.

Bottom Line: We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally.Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3.This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia.

ABSTRACT
Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

Show MeSH