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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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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.
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fig1: 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.

Mentions: The Gli3-PHS part (residues 1–673) was shown to contain gene repressor activity [3]. Using a two-hybrid screening technology it was shown that almost the same part (residues 1–613) in Gli3 binds to Ski [9]. The Ski binding site on Gli3 was determined to the region from residue 152 to 397 using pull-down assays [9]. Since Ski is known to be part of a gene repressor complex including HDAC, it was suggested that Gli3 exerts its repressor activity through binding of Ski and recruitment of HDAC [9]. However, most of the Ski binding region is conserved between all three Gli proteins, suggesting that Ski binding and HDAC recruitment is part of a general transcription termination signal common to all Gli proteins. Likewise, the SUFU binding site (BS) on Gli proteins (SYGH) is also found in all three Gli proteins [10]. Also SUFU is known to recruit HDAC through recruitment of SAP18 and Sin3A [11] and therefore, SUFU binding may also be regarded as a general mechanism to turn off Gli mediated transcription. Recently, two sites in Gli1 were identified as responsible for protein degradation [12]. One peptide (degron) was in the C-terminal part (DC) whereas the other was found in the N-terminal part (DN). In fact the DN peptide is located very close to the SUFU binding site and is conserved also in Gli2 and Gli3. A previous study identified the peptide 94–280 of Gli2 as a repressor part and removal of a corresponding part in Gli3 (residues 1–344) had strong positive effect on transcription [13]. In contrast, when this part of Gli1 (residues 1–134) was removed there was no effect on transcription as compared to wild type protein [13]. The last approximately 100 residues of this region is conserved between the three Gli proteins, and those are the parts that overlap with the identified Ski binding part, contain DN and the SYGH peptide. Fig. 1A shows a schematic alignment of the N-terminal parts of mammalian Gli proteins, until the end of the Zn-fingers (corresponding to the PHS-domain), with indications of the respective domains describe above. From this work and a previous paper [3] it is suggested that the gene repressor function is localized to the N-terminal (grey) part of Gli2 and Gli3. The physiological significance was shown by regulation of PTCH1 transcription [3]. We suggest that the other mechanisms with negative gene transcription activity (Ski BS, SUFU BS and DN peptide) that are common to all Gli proteins, are general means to terminate Gli induced transcription. At least two of these pathways (Ski and SUFU through interaction with SAP18 and Sin3A) probably recruit HDACs to terminate transcription and increase the degradation of the Gli protein.


Identification of the gene transcription repressor domain of Gli3.

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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.
Mentions: The Gli3-PHS part (residues 1–673) was shown to contain gene repressor activity [3]. Using a two-hybrid screening technology it was shown that almost the same part (residues 1–613) in Gli3 binds to Ski [9]. The Ski binding site on Gli3 was determined to the region from residue 152 to 397 using pull-down assays [9]. Since Ski is known to be part of a gene repressor complex including HDAC, it was suggested that Gli3 exerts its repressor activity through binding of Ski and recruitment of HDAC [9]. However, most of the Ski binding region is conserved between all three Gli proteins, suggesting that Ski binding and HDAC recruitment is part of a general transcription termination signal common to all Gli proteins. Likewise, the SUFU binding site (BS) on Gli proteins (SYGH) is also found in all three Gli proteins [10]. Also SUFU is known to recruit HDAC through recruitment of SAP18 and Sin3A [11] and therefore, SUFU binding may also be regarded as a general mechanism to turn off Gli mediated transcription. Recently, two sites in Gli1 were identified as responsible for protein degradation [12]. One peptide (degron) was in the C-terminal part (DC) whereas the other was found in the N-terminal part (DN). In fact the DN peptide is located very close to the SUFU binding site and is conserved also in Gli2 and Gli3. A previous study identified the peptide 94–280 of Gli2 as a repressor part and removal of a corresponding part in Gli3 (residues 1–344) had strong positive effect on transcription [13]. In contrast, when this part of Gli1 (residues 1–134) was removed there was no effect on transcription as compared to wild type protein [13]. The last approximately 100 residues of this region is conserved between the three Gli proteins, and those are the parts that overlap with the identified Ski binding part, contain DN and the SYGH peptide. Fig. 1A shows a schematic alignment of the N-terminal parts of mammalian Gli proteins, until the end of the Zn-fingers (corresponding to the PHS-domain), with indications of the respective domains describe above. From this work and a previous paper [3] it is suggested that the gene repressor function is localized to the N-terminal (grey) part of Gli2 and Gli3. The physiological significance was shown by regulation of PTCH1 transcription [3]. We suggest that the other mechanisms with negative gene transcription activity (Ski BS, SUFU BS and DN peptide) that are common to all Gli proteins, are general means to terminate Gli induced transcription. At least two of these pathways (Ski and SUFU through interaction with SAP18 and Sin3A) probably recruit HDACs to terminate transcription and increase the degradation of the Gli protein.

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