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

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.

Figure 1.

ChIP-seq analysis for FoxO1 binding in hepatic chromatin. (A) Total ChIP-seq reads are shown in WT or l-FoxO1 KO (KO) chromatin enriched with an anti-FoxO1 or IgG. (B) Peaks were found by SEAN, as described in ‘Supplementary Materials and Methods S1’. FoxO1-binding peaks in WT FoxO1/WT IgG (FoxO1/IgG) were compared with those of WT FoxO1/KO FoxO1 (WT/KO) and overlapping peaks were identified.

Figure 2.

Representative view of a ChIP-seq peak. FoxO1-binding peaks were mapped onto the mouse reference genome mm9 and visualized using the UCSC Genome Browser. Arrows indicate peaks associated with genes: (A), Elk4; (B), Pck1; (C), Pdk4. WT = wild-type; KO = l-FoxO1 KO; Fx = FoxO1.

Figure 3.

Density map plots of FoxO1 ChIP-seq peaks. (A) The top panel represents the read distribution of peaks in ChIP-seq for FoxO1, whereas the bottom panel represents the read distribution of random peaks. (B) The top and the bottom plots represent cumulative reads of FoxO1 ChIP-seq peaks and those of random peaks, respectively. The maximum number of read is set to 100.

Figure 4.

Genome-wide distribution of FoxO1-binding sites. (A) Genomic distribution of FoxO1-binding sites relative to mouse RefSeq genes is shown. The promoter regions are defined as 2 kb of 5′ flanking sequence. (B) Peak distribution relative TSS is shown. The peak distribution of FoxO1 binding is shown in red, whereas the distribution of random peaks is shown in blue. (C) The top-scoring motif present in the FoxO1-binding peaks is shown by MEME motif analysis. E = 3.1e-150.

Figure 5.

Gene set enrichment analysis. All genes (n = ∼2.5 × 10⁴) in microarray expression profiling from fed versus fasted mouse livers were ranked by fold difference and P-value and expressed on the x-axis. This data set was compared with the gene list of nearest genes (n = 296) identified in ChIP-seq peaks located within 20 kb of a known gene. The plot of running sum for genes in ChIP-seq data is shown at the bottom with the leading-edge subset. The location of maximum enrichment score indicates a high correlation of the two data sets.

Figure 6.

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.