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Genome-wide analysis of FoxO1 binding in hepatic chromatin: potential involvement of FoxO1 in linking retinoid signaling to hepatic gluconeogenesis.

Shin DJ, Joshi P, Hong SH, Mosure K, Shin DG, Osborne TF - Nucleic Acids Res. (2012)

Bottom Line: We show here that FoxO1 directly binds to the genomic sites for the genes in retinoid metabolism.Notably, deletion of FoxO1 caused a significantly reduced induction of Pck1 and Pdk4 in response to retinoids.As Pck1 and Pdk4 are downstream targets of retinoid signaling, these results suggest that FoxO1 plays a potential role in linking retinoid metabolism to hepatic gluconeogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA. djshin@sanfordburnham.org

ABSTRACT
The forkhead transcription factor FoxO1 is a critical regulator of hepatic glucose and lipid metabolism, and dysregulation of FoxO1 function has been implicated in diabetes and insulin resistance. We globally identified FoxO1 occupancy in mouse hepatic chromatin on a genome-wide level by chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq). To establish the specific functional significance of FoxO1 against other FoxO proteins, ChIP-seq was performed with chromatin from liver-specific FoxO1 knockout and wild-type mice. Here we identified 401 genome-wide FoxO1-binding locations. Motif search reveals a sequence element, 5' GTAAACA 3', consistent with a previously known FoxO1-binding site. Gene set enrichment analysis shows that the data from FoxO1 ChIP-seq are highly correlated with the global expression profiling of genes regulated by FoxO1, demonstrating the functional relevance of our FoxO1 ChIP-seq study. Interestingly, gene ontology analysis reveals the functional significance of FoxO1 in retinoid metabolic processes. We show here that FoxO1 directly binds to the genomic sites for the genes in retinoid metabolism. Notably, deletion of FoxO1 caused a significantly reduced induction of Pck1 and Pdk4 in response to retinoids. As Pck1 and Pdk4 are downstream targets of retinoid signaling, these results suggest that FoxO1 plays a potential role in linking retinoid metabolism to hepatic gluconeogenesis.

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FoxO1 is required for full response of Pck1 and Pdk4 to retinoids. Primary mouse hepatocytes were infected with an adenovirus expressing FoxO1 shRNA or un-specific RNAi for 24 h. Hepatocytes were treated with retinol (20 µM) or atRA (20 µM) for 6 h. mRNA levels were measured by qPCR and were normalized for ribosomal protein Rpl32 mRNA levels. Results are expressed as fold change relative to those of control. The mean values obtained from triplicates in each group as shown with error bars. *P < 0.05. Ctrl = control; rol = retinol; atRA = all-trans RA.
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gks932-F6: FoxO1 is required for full response of Pck1 and Pdk4 to retinoids. Primary mouse hepatocytes were infected with an adenovirus expressing FoxO1 shRNA or un-specific RNAi for 24 h. Hepatocytes were treated with retinol (20 µM) or atRA (20 µM) for 6 h. mRNA levels were measured by qPCR and were normalized for ribosomal protein Rpl32 mRNA levels. Results are expressed as fold change relative to those of control. The mean values obtained from triplicates in each group as shown with error bars. *P < 0.05. Ctrl = control; rol = retinol; atRA = all-trans RA.

Mentions: If FoxO1 is an important player in retinoid metabolism, deletion of FoxO1 should result in impaired retinoid signaling in regulating its downstream signaling targets. To test this idea, we knock-downed FoxO1 in primary hepatocytes by an shRNA adenovirus targeting FoxO1 (FoxO1 shRNA) and analysed mRNA expression of genes for Pck1 and Pdk4 in response to retinol or atRA, as Pck1 and Pdk4 have been shown regulated by retinoids (13,14,16–18). Efficient knockdown of FoxO1 by FoxO1 shRNA is shown in Supplementary Figure S5 along with significantly reduced expression of Pck1, a known FoxO1 target. In line with our ChIP-seq analysis, the expression levels of genes for Retsat, Rbp1, Dhrs9 and Rdh8 in retinoid metabolism were significantly lower in hepatocytes where FoxO1 expression was disrupted. As shown in Figure 6, expression of Pck1 was significantly induced by retinol and atRA, consistent with the presence of retinoic acid response elements in the promoter region (13,16). PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex, promoting the conversion of pyruvate to lactate that is used for hepatic gluconeogenesis. We also observed markedly increased Pdk4 expression by retinol and atRA. In contrast, the gene encoding glucose-6-phosphatase showed no significant response to retinol and atRA. Notably, deletion of FoxO1 resulted in a significantly blunted induction of Pck1 and Pdk4 in response to retinol and atRA, suggesting that FoxO1 is required for full response of the genes to retinoids.Figure 6.


Genome-wide analysis of FoxO1 binding in hepatic chromatin: potential involvement of FoxO1 in linking retinoid signaling to hepatic gluconeogenesis.

Shin DJ, Joshi P, Hong SH, Mosure K, Shin DG, Osborne TF - Nucleic Acids Res. (2012)

FoxO1 is required for full response of Pck1 and Pdk4 to retinoids. Primary mouse hepatocytes were infected with an adenovirus expressing FoxO1 shRNA or un-specific RNAi for 24 h. Hepatocytes were treated with retinol (20 µM) or atRA (20 µM) for 6 h. mRNA levels were measured by qPCR and were normalized for ribosomal protein Rpl32 mRNA levels. Results are expressed as fold change relative to those of control. The mean values obtained from triplicates in each group as shown with error bars. *P < 0.05. Ctrl = control; rol = retinol; atRA = all-trans RA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526316&req=5

gks932-F6: FoxO1 is required for full response of Pck1 and Pdk4 to retinoids. Primary mouse hepatocytes were infected with an adenovirus expressing FoxO1 shRNA or un-specific RNAi for 24 h. Hepatocytes were treated with retinol (20 µM) or atRA (20 µM) for 6 h. mRNA levels were measured by qPCR and were normalized for ribosomal protein Rpl32 mRNA levels. Results are expressed as fold change relative to those of control. The mean values obtained from triplicates in each group as shown with error bars. *P < 0.05. Ctrl = control; rol = retinol; atRA = all-trans RA.
Mentions: If FoxO1 is an important player in retinoid metabolism, deletion of FoxO1 should result in impaired retinoid signaling in regulating its downstream signaling targets. To test this idea, we knock-downed FoxO1 in primary hepatocytes by an shRNA adenovirus targeting FoxO1 (FoxO1 shRNA) and analysed mRNA expression of genes for Pck1 and Pdk4 in response to retinol or atRA, as Pck1 and Pdk4 have been shown regulated by retinoids (13,14,16–18). Efficient knockdown of FoxO1 by FoxO1 shRNA is shown in Supplementary Figure S5 along with significantly reduced expression of Pck1, a known FoxO1 target. In line with our ChIP-seq analysis, the expression levels of genes for Retsat, Rbp1, Dhrs9 and Rdh8 in retinoid metabolism were significantly lower in hepatocytes where FoxO1 expression was disrupted. As shown in Figure 6, expression of Pck1 was significantly induced by retinol and atRA, consistent with the presence of retinoic acid response elements in the promoter region (13,16). PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex, promoting the conversion of pyruvate to lactate that is used for hepatic gluconeogenesis. We also observed markedly increased Pdk4 expression by retinol and atRA. In contrast, the gene encoding glucose-6-phosphatase showed no significant response to retinol and atRA. Notably, deletion of FoxO1 resulted in a significantly blunted induction of Pck1 and Pdk4 in response to retinol and atRA, suggesting that FoxO1 is required for full response of the genes to retinoids.Figure 6.

Bottom Line: We show here that FoxO1 directly binds to the genomic sites for the genes in retinoid metabolism.Notably, deletion of FoxO1 caused a significantly reduced induction of Pck1 and Pdk4 in response to retinoids.As Pck1 and Pdk4 are downstream targets of retinoid signaling, these results suggest that FoxO1 plays a potential role in linking retinoid metabolism to hepatic gluconeogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA. djshin@sanfordburnham.org

ABSTRACT
The forkhead transcription factor FoxO1 is a critical regulator of hepatic glucose and lipid metabolism, and dysregulation of FoxO1 function has been implicated in diabetes and insulin resistance. We globally identified FoxO1 occupancy in mouse hepatic chromatin on a genome-wide level by chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq). To establish the specific functional significance of FoxO1 against other FoxO proteins, ChIP-seq was performed with chromatin from liver-specific FoxO1 knockout and wild-type mice. Here we identified 401 genome-wide FoxO1-binding locations. Motif search reveals a sequence element, 5' GTAAACA 3', consistent with a previously known FoxO1-binding site. Gene set enrichment analysis shows that the data from FoxO1 ChIP-seq are highly correlated with the global expression profiling of genes regulated by FoxO1, demonstrating the functional relevance of our FoxO1 ChIP-seq study. Interestingly, gene ontology analysis reveals the functional significance of FoxO1 in retinoid metabolic processes. We show here that FoxO1 directly binds to the genomic sites for the genes in retinoid metabolism. Notably, deletion of FoxO1 caused a significantly reduced induction of Pck1 and Pdk4 in response to retinoids. As Pck1 and Pdk4 are downstream targets of retinoid signaling, these results suggest that FoxO1 plays a potential role in linking retinoid metabolism to hepatic gluconeogenesis.

Show MeSH
Related in: MedlinePlus