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HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity.

ZeRuth GT, Williams JG, Cole YC, Jetten AM - PLoS ONE (2015)

Bottom Line: However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation.Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity.This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

No MeSH data available.


Related in: MedlinePlus

Itch inhibits Glis3-mediated transactivation of target genes.A. INS1 832/13 cells were transfected with pGL4.27 or p3xGlisBS-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, cells were assayed for luciferase and β-galactosidase activity and the normalized relative luciferase activity (nRLU) was calculated and plotted. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. B. HEK293T cells were transfected with p-mIP-696-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h cells were assayed as described for A. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. C. INS1 832/13 cells were transfected with Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, RNA was collected and rIns2 mRNA was measured by qRT-PCR analysis. Each bar represents relative Ins2 mRNA normalized to 18s rRNA +/- SEM. * Indicates statistically different value compared to empty vector control p < 0.02.
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pone.0131303.g008: Itch inhibits Glis3-mediated transactivation of target genes.A. INS1 832/13 cells were transfected with pGL4.27 or p3xGlisBS-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, cells were assayed for luciferase and β-galactosidase activity and the normalized relative luciferase activity (nRLU) was calculated and plotted. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. B. HEK293T cells were transfected with p-mIP-696-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h cells were assayed as described for A. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. C. INS1 832/13 cells were transfected with Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, RNA was collected and rIns2 mRNA was measured by qRT-PCR analysis. Each bar represents relative Ins2 mRNA normalized to 18s rRNA +/- SEM. * Indicates statistically different value compared to empty vector control p < 0.02.

Mentions: Because Glis3 functions as a transcriptional activator for several target genes, including insulin, we were interested in determining whether Itch had any effect on Glis3-mediated transcriptional activation. Itch, but not the catalytically inactive mutant inhibited the transactivation of a Luc reporter driven by 3 tandem copies of the GlisBS in INS1 832/13 pancreatic β cells (Fig 8A). Importantly, Itch expression reduced activation of the reporter both in the presence or absence of exogenous Glis3 suggesting that it likely promotes the degradation of endogenous Glis3 in INS1 832/13 cells. Consistent with these results, luciferase reporter assays performed in HEK293T cells using the mouse Ins2 promoter driven reporter construct, p-mIP-696-Luc, demonstrated that exogenous Itch expression again significantly reduced Glis3-mediated reporter activation (Fig 8B). Activation of the Ins2 reporter by Glis3 was not inhibited by co-expression of the catalytically inactive Itch C832G mutant. Moreover, Itch did not inhibit the transactivation capacity of the Glis3 PY461 mutant, which was stable in the presence of exogenous Itch expression. Together, these data indicate that the ligase activity of Itch as well as its interaction with the PY motif of Glis3 is required for the inhibition of Glis3 transcriptional activation and that the decreased level of activation is at least in part due to reduced protein levels following Itch-mediated degradation of Glis3. It further indicates that the effect of Itch is specific for Glis3 and not due to an effect on the general transcriptional machinery. In order to determine whether Itch had a similar effect on endogenous insulin transcription in INS1 cells, Myc-Itch or Myc-Itch C832G was transiently transfected into INS1 832/13 cells and their effect on rIns2 mRNA expression examined by QRT-PCR. Indeed, expression of Itch resulted in a > 60% decrease in Ins2 mRNA, whereas Itch C832G expression had little effect (Fig 8C). In contrast, Smurf2 or NEDD4 expression did not significantly alter the activation of rIns2 transcription by Glis3 (S4A and S4B Fig).


HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity.

ZeRuth GT, Williams JG, Cole YC, Jetten AM - PLoS ONE (2015)

Itch inhibits Glis3-mediated transactivation of target genes.A. INS1 832/13 cells were transfected with pGL4.27 or p3xGlisBS-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, cells were assayed for luciferase and β-galactosidase activity and the normalized relative luciferase activity (nRLU) was calculated and plotted. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. B. HEK293T cells were transfected with p-mIP-696-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h cells were assayed as described for A. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. C. INS1 832/13 cells were transfected with Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, RNA was collected and rIns2 mRNA was measured by qRT-PCR analysis. Each bar represents relative Ins2 mRNA normalized to 18s rRNA +/- SEM. * Indicates statistically different value compared to empty vector control p < 0.02.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493090&req=5

pone.0131303.g008: Itch inhibits Glis3-mediated transactivation of target genes.A. INS1 832/13 cells were transfected with pGL4.27 or p3xGlisBS-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, cells were assayed for luciferase and β-galactosidase activity and the normalized relative luciferase activity (nRLU) was calculated and plotted. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. B. HEK293T cells were transfected with p-mIP-696-Luc, FLAG-Glis3, the PY461 mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h cells were assayed as described for A. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. C. INS1 832/13 cells were transfected with Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, RNA was collected and rIns2 mRNA was measured by qRT-PCR analysis. Each bar represents relative Ins2 mRNA normalized to 18s rRNA +/- SEM. * Indicates statistically different value compared to empty vector control p < 0.02.
Mentions: Because Glis3 functions as a transcriptional activator for several target genes, including insulin, we were interested in determining whether Itch had any effect on Glis3-mediated transcriptional activation. Itch, but not the catalytically inactive mutant inhibited the transactivation of a Luc reporter driven by 3 tandem copies of the GlisBS in INS1 832/13 pancreatic β cells (Fig 8A). Importantly, Itch expression reduced activation of the reporter both in the presence or absence of exogenous Glis3 suggesting that it likely promotes the degradation of endogenous Glis3 in INS1 832/13 cells. Consistent with these results, luciferase reporter assays performed in HEK293T cells using the mouse Ins2 promoter driven reporter construct, p-mIP-696-Luc, demonstrated that exogenous Itch expression again significantly reduced Glis3-mediated reporter activation (Fig 8B). Activation of the Ins2 reporter by Glis3 was not inhibited by co-expression of the catalytically inactive Itch C832G mutant. Moreover, Itch did not inhibit the transactivation capacity of the Glis3 PY461 mutant, which was stable in the presence of exogenous Itch expression. Together, these data indicate that the ligase activity of Itch as well as its interaction with the PY motif of Glis3 is required for the inhibition of Glis3 transcriptional activation and that the decreased level of activation is at least in part due to reduced protein levels following Itch-mediated degradation of Glis3. It further indicates that the effect of Itch is specific for Glis3 and not due to an effect on the general transcriptional machinery. In order to determine whether Itch had a similar effect on endogenous insulin transcription in INS1 cells, Myc-Itch or Myc-Itch C832G was transiently transfected into INS1 832/13 cells and their effect on rIns2 mRNA expression examined by QRT-PCR. Indeed, expression of Itch resulted in a > 60% decrease in Ins2 mRNA, whereas Itch C832G expression had little effect (Fig 8C). In contrast, Smurf2 or NEDD4 expression did not significantly alter the activation of rIns2 transcription by Glis3 (S4A and S4B Fig).

Bottom Line: However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation.Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity.This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

No MeSH data available.


Related in: MedlinePlus