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Control of Foxo1 gene expression by co-activator P300.

Wondisford AR, Xiong L, Chang E, Meng S, Meyers DJ, Li M, Cole PA, He L - J. Biol. Chem. (2013)

Bottom Line: In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation.Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs.In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Metabolism, Department of Pediatrics and.

ABSTRACT
FOXO1 is an important downstream mediator of the insulin signaling pathway. In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation. A reduction in nuclear FOXO1 levels then leads to suppression of hepatic glucose production. However, the mechanism leading to expression of Foxo1 gene in the fasted state is less clear. We found that Foxo1 mRNA and FOXO1 protein levels of Foxo1 were increased significantly in the liver of mice after 16 h of fasting. Furthermore, dibutyrl cAMP stimulated the expression of Foxo1 at both mRNA and protein level in hepatocytes. Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs. Interestingly, only depletion of co-activator P300 resulted in the decrease of Foxo1 mRNA and FOXO1 protein levels. In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels. By characterization of Foxo1 gene promoter, P300 regulates the Foxo1 gene expression through the binding to tandem cAMP-response element sites in the proximal promoter region of Foxo1 gene.

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P300 binds to the proximal promoter region of the Foxo1 gene.a, PKA stimulated the binding of CREB and p300 to CREs in the proximal promoter of the Foxo1 gene. 48 h after the transfection of PKA expression plasmid, Hepa1–6 cells were fixed with formaldehyde followed by immunoprecipitation with CREB and P300 antibodies. The DNA in the immunoprecipitates was amplified using primers encompassing 150 bp including the CREs in the promoter of the mouse Foxo1 gene (n = 3). b, Hepa1–6 cells were transfected with PKA or together with HA-tagged P300 expression plasmid, followed by the fixation and immunoprecipitation using anti-HA tag-specific antibody. The binding of HA-tagged P300 was determined by real-time PCR (n = 3). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.
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Figure 6: P300 binds to the proximal promoter region of the Foxo1 gene.a, PKA stimulated the binding of CREB and p300 to CREs in the proximal promoter of the Foxo1 gene. 48 h after the transfection of PKA expression plasmid, Hepa1–6 cells were fixed with formaldehyde followed by immunoprecipitation with CREB and P300 antibodies. The DNA in the immunoprecipitates was amplified using primers encompassing 150 bp including the CREs in the promoter of the mouse Foxo1 gene (n = 3). b, Hepa1–6 cells were transfected with PKA or together with HA-tagged P300 expression plasmid, followed by the fixation and immunoprecipitation using anti-HA tag-specific antibody. The binding of HA-tagged P300 was determined by real-time PCR (n = 3). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.

Mentions: To investigate whether recruitment of CREB and P300 to CREs was induced by PKA stimulation, we conducted a ChIP assay. Hepa1–6 cells were transfected with control or PKA-containing plasmids to assess the occupancy of CREB and P300 on Foxo1 and a negative control Gapdh promoter. Fig. 6a shows the quantitative PCR for Foxo1 or the negative control Gapdh promoter of either CREB- or P300-immunopreciptated chromatin. PKA transfection markedly increased the occupancy of both CREB and P300 on the Foxo1 promoter but not on the negative control Gapdh promoter. These data correlate with the functional data obtained in the same mouse hepatocyte cultures as shown in Fig. 5. To probe further whether PKA stimulated the occupancy of P300 on the proximal Foxo1 promoter, we overexpressed HA-tagged P300 together with PKA and used a HA tag-specific antibody to immunoprecipitate the cross-linked Foxo1 promoter in a ChIP assay. The binding of HA-tagged P300 increased >6-fold in the presence of PKA stimulation (Fig. 6b).


Control of Foxo1 gene expression by co-activator P300.

Wondisford AR, Xiong L, Chang E, Meng S, Meyers DJ, Li M, Cole PA, He L - J. Biol. Chem. (2013)

P300 binds to the proximal promoter region of the Foxo1 gene.a, PKA stimulated the binding of CREB and p300 to CREs in the proximal promoter of the Foxo1 gene. 48 h after the transfection of PKA expression plasmid, Hepa1–6 cells were fixed with formaldehyde followed by immunoprecipitation with CREB and P300 antibodies. The DNA in the immunoprecipitates was amplified using primers encompassing 150 bp including the CREs in the promoter of the mouse Foxo1 gene (n = 3). b, Hepa1–6 cells were transfected with PKA or together with HA-tagged P300 expression plasmid, followed by the fixation and immunoprecipitation using anti-HA tag-specific antibody. The binding of HA-tagged P300 was determined by real-time PCR (n = 3). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: P300 binds to the proximal promoter region of the Foxo1 gene.a, PKA stimulated the binding of CREB and p300 to CREs in the proximal promoter of the Foxo1 gene. 48 h after the transfection of PKA expression plasmid, Hepa1–6 cells were fixed with formaldehyde followed by immunoprecipitation with CREB and P300 antibodies. The DNA in the immunoprecipitates was amplified using primers encompassing 150 bp including the CREs in the promoter of the mouse Foxo1 gene (n = 3). b, Hepa1–6 cells were transfected with PKA or together with HA-tagged P300 expression plasmid, followed by the fixation and immunoprecipitation using anti-HA tag-specific antibody. The binding of HA-tagged P300 was determined by real-time PCR (n = 3). Asterisk (*) signifies that groups with same treatment are significantly different (p < 0.05). Error bars, S.D.
Mentions: To investigate whether recruitment of CREB and P300 to CREs was induced by PKA stimulation, we conducted a ChIP assay. Hepa1–6 cells were transfected with control or PKA-containing plasmids to assess the occupancy of CREB and P300 on Foxo1 and a negative control Gapdh promoter. Fig. 6a shows the quantitative PCR for Foxo1 or the negative control Gapdh promoter of either CREB- or P300-immunopreciptated chromatin. PKA transfection markedly increased the occupancy of both CREB and P300 on the Foxo1 promoter but not on the negative control Gapdh promoter. These data correlate with the functional data obtained in the same mouse hepatocyte cultures as shown in Fig. 5. To probe further whether PKA stimulated the occupancy of P300 on the proximal Foxo1 promoter, we overexpressed HA-tagged P300 together with PKA and used a HA tag-specific antibody to immunoprecipitate the cross-linked Foxo1 promoter in a ChIP assay. The binding of HA-tagged P300 increased >6-fold in the presence of PKA stimulation (Fig. 6b).

Bottom Line: In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation.Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs.In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Metabolism, Department of Pediatrics and.

ABSTRACT
FOXO1 is an important downstream mediator of the insulin signaling pathway. In the fed state, elevated insulin phosphorylates FOXO1 via AKT, leading to its nuclear exclusion and degradation. A reduction in nuclear FOXO1 levels then leads to suppression of hepatic glucose production. However, the mechanism leading to expression of Foxo1 gene in the fasted state is less clear. We found that Foxo1 mRNA and FOXO1 protein levels of Foxo1 were increased significantly in the liver of mice after 16 h of fasting. Furthermore, dibutyrl cAMP stimulated the expression of Foxo1 at both mRNA and protein level in hepatocytes. Because cAMP-PKA regulates hepatic glucose production through cAMP-response element-binding protein co-activators, we depleted these co-activators using adenoviral shRNAs. Interestingly, only depletion of co-activator P300 resulted in the decrease of Foxo1 mRNA and FOXO1 protein levels. In addition, inhibition of histone acetyltransferase activity of P300 significantly decreased hepatic Foxo1 mRNA and FOXO1 protein levels in fasted mice, as well as fasting blood glucose levels. By characterization of Foxo1 gene promoter, P300 regulates the Foxo1 gene expression through the binding to tandem cAMP-response element sites in the proximal promoter region of Foxo1 gene.

Show MeSH
Related in: MedlinePlus