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. 2009 Jan 5;583(1):224-8.
doi: 10.1016/j.febslet.2008.12.010. Epub 2008 Dec 10.

Identification of the gene transcription repressor domain of Gli3

Robert Tsanev  1 , Piret Tiigimägi, Piret Michelson, Madis Metsis, Torben Østerlund, Priit Kogerman
Affiliations

Identification of the gene transcription repressor domain of Gli3

Robert Tsanev et al. FEBS Lett. .

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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Figures

Fig. 1
Fig. 1
Sequence analyzes of the N-terminal halves of Gli1, Gli2 and Gli3, as well as analyzes of the transcriptional regulation by Gli1 and Gli3 and the PHS domain of Gli3. (A) Schematic alignment of Gli1, Gli2 and Gli3 in the N-terminal half until the end of the Zn-finger DBD (Zn-finger; lined). The suggested repressor domain (Rep Dom; grey) is only found in Gli2 and Gli3. The sequences for the Sufu binding site (Sufu BS) and DegronN (Degron N) is found in all three Gli proteins (hatched and black, respectively). Above is a line indicating the presumed Ski binding region (Ski BS). Both Ski and Sufu are likely to recruit HDACs through Sin3A and induce transcriptional termination by this mechanism. (B) HEK293 cells were transfected with a Gli responsive luciferase reporter and Gli1, Gli3 or N-terminal parts of these corresponding to the PHS domain, or combinations to assess the effects of these on transcription. Error bars indicate the standard deviations of triplicate analyzes.
Fig. 2
Fig. 2
Analyzes of the repressor function of the Gli3-PHS domain and the repressor domain (RD) in Shh-L2 cells. Shh-L2 cells (with an incorporated Gli responsive luciferase reporter gene) were transfected with Gli1, Gli3, Gli3-PHS, Gli3ΔRD, Gli3-PHSΔRD or combinations of these to assess the effect on transcription alone or on the Gli1 induced transcription. The analyzes were performed at least three times and error bars indicate the standard deviations.
Fig. 3
Fig. 3
Determination of the minimal repressor domain of Gli3. The repressor domain (residues 105–246) or parts of this were expressed together with the DBD of Gal4 and assessed for repression of Gal4 induced transcription in HEK293 cells (mock). Also a larger part of the Gli3 N-terminal part was measured since this is known to have significant repressor function (residues 1–480). The analyzes were performed three to five times and error bars show the standard deviations.
Fig. 4
Fig. 4
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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