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Gli2 acetylation at lysine 757 regulates hedgehog-dependent transcriptional output by preventing its promoter occupancy.

Coni S, Antonucci L, D'Amico D, Di Magno L, Infante P, De Smaele E, Giannini G, Di Marcotullio L, Screpanti I, Gulino A, Canettieri G - PLoS ONE (2013)

Bottom Line: Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling.Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin.Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 7277, Inserm 1091, Institut de Biologie Valrose (iBV), Centre de Biochimie, Nice, France.

ABSTRACT
The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.

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Removal of Gli2 acetylation enhances Hh-dependent transcription.(A) K757 acetylation of wild type Myc tagged Gli2 and K757R mutant in HEK293T cells. Cells extract were immunoprecipitated with anti-Myc antibody and acetylation was detected by anti acetyl-Gli2(Lys757) specific antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. (B) Acetylation of endogenous Gli2 in NIH3T3 cells, treated with SAG or DMSO for 24 hours. Acetylation was detected with anti acetyl-Gli2(Lys757) specific antisera. (C) (Top) Luciferase assay in NIH3T3 cells transfected with 8× Gli-Luc reporter, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Gli1 mRNA levels normalized with the housekeeping HPRT mRNA in NIH3T3 cells transfected with Myc-Gli2 WT and K757R mutant. (D) Transcriptional activity of Myc-Gli2 WT and K757R mutant in response to SAG treatment: Ptch1 mRNA levels (QPCR), normalized with the housekeeping GAPDH mRNA in 24 hours SAG-treated NIH3T3 cells, transfected with Empty vector, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty, **p<0.05 vs DMSO; Results are shown as the average ± SD of triplicate experiments (n = 3).
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pone-0065718-g002: Removal of Gli2 acetylation enhances Hh-dependent transcription.(A) K757 acetylation of wild type Myc tagged Gli2 and K757R mutant in HEK293T cells. Cells extract were immunoprecipitated with anti-Myc antibody and acetylation was detected by anti acetyl-Gli2(Lys757) specific antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. (B) Acetylation of endogenous Gli2 in NIH3T3 cells, treated with SAG or DMSO for 24 hours. Acetylation was detected with anti acetyl-Gli2(Lys757) specific antisera. (C) (Top) Luciferase assay in NIH3T3 cells transfected with 8× Gli-Luc reporter, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Gli1 mRNA levels normalized with the housekeeping HPRT mRNA in NIH3T3 cells transfected with Myc-Gli2 WT and K757R mutant. (D) Transcriptional activity of Myc-Gli2 WT and K757R mutant in response to SAG treatment: Ptch1 mRNA levels (QPCR), normalized with the housekeeping GAPDH mRNA in 24 hours SAG-treated NIH3T3 cells, transfected with Empty vector, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty, **p<0.05 vs DMSO; Results are shown as the average ± SD of triplicate experiments (n = 3).

Mentions: Toward this end, we generated a rabbit polyclonal antibody against the acetylated K757 of Gli2, which was able to readily detect this modification and did not react against K757R mutant Gli2 (Fig. 2A).


Gli2 acetylation at lysine 757 regulates hedgehog-dependent transcriptional output by preventing its promoter occupancy.

Coni S, Antonucci L, D'Amico D, Di Magno L, Infante P, De Smaele E, Giannini G, Di Marcotullio L, Screpanti I, Gulino A, Canettieri G - PLoS ONE (2013)

Removal of Gli2 acetylation enhances Hh-dependent transcription.(A) K757 acetylation of wild type Myc tagged Gli2 and K757R mutant in HEK293T cells. Cells extract were immunoprecipitated with anti-Myc antibody and acetylation was detected by anti acetyl-Gli2(Lys757) specific antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. (B) Acetylation of endogenous Gli2 in NIH3T3 cells, treated with SAG or DMSO for 24 hours. Acetylation was detected with anti acetyl-Gli2(Lys757) specific antisera. (C) (Top) Luciferase assay in NIH3T3 cells transfected with 8× Gli-Luc reporter, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Gli1 mRNA levels normalized with the housekeeping HPRT mRNA in NIH3T3 cells transfected with Myc-Gli2 WT and K757R mutant. (D) Transcriptional activity of Myc-Gli2 WT and K757R mutant in response to SAG treatment: Ptch1 mRNA levels (QPCR), normalized with the housekeeping GAPDH mRNA in 24 hours SAG-treated NIH3T3 cells, transfected with Empty vector, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty, **p<0.05 vs DMSO; Results are shown as the average ± SD of triplicate experiments (n = 3).
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pone-0065718-g002: Removal of Gli2 acetylation enhances Hh-dependent transcription.(A) K757 acetylation of wild type Myc tagged Gli2 and K757R mutant in HEK293T cells. Cells extract were immunoprecipitated with anti-Myc antibody and acetylation was detected by anti acetyl-Gli2(Lys757) specific antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. (B) Acetylation of endogenous Gli2 in NIH3T3 cells, treated with SAG or DMSO for 24 hours. Acetylation was detected with anti acetyl-Gli2(Lys757) specific antisera. (C) (Top) Luciferase assay in NIH3T3 cells transfected with 8× Gli-Luc reporter, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Gli1 mRNA levels normalized with the housekeeping HPRT mRNA in NIH3T3 cells transfected with Myc-Gli2 WT and K757R mutant. (D) Transcriptional activity of Myc-Gli2 WT and K757R mutant in response to SAG treatment: Ptch1 mRNA levels (QPCR), normalized with the housekeeping GAPDH mRNA in 24 hours SAG-treated NIH3T3 cells, transfected with Empty vector, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty, **p<0.05 vs DMSO; Results are shown as the average ± SD of triplicate experiments (n = 3).
Mentions: Toward this end, we generated a rabbit polyclonal antibody against the acetylated K757 of Gli2, which was able to readily detect this modification and did not react against K757R mutant Gli2 (Fig. 2A).

Bottom Line: Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling.Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin.Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 7277, Inserm 1091, Institut de Biologie Valrose (iBV), Centre de Biochimie, Nice, France.

ABSTRACT
The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.

Show MeSH
Related in: MedlinePlus