HNF4 Regulates Fatty Acid Oxidation and Is Required for Renewal of Intestinal Stem Cells in Mice

Abstract

Background & aims: Functions of intestinal stem cells (ISCs) are regulated by diet and metabolic pathways. Hepatocyte nuclear factor 4 (HNF4) family are transcription factors that bind fatty acids. We investigated how HNF4 transcription factors regulate metabolism and their functions in ISCs in mice.

Methods: We performed studies with Villin-Cre^ERT2;Lgr5-EGFP-IRES-Cre^ERT2;Hnf4α^f/f;Hnf4γ^{Crispr/Crispr} mice, hereafter referred to Hnf4αγ^DKO. Mice were given tamoxifen to induce Cre recombinase. Mice transgenic with only Cre alleles (Villin-Cre^ERT2, Lgr5-EGFP-IRES-Cre^ERT2, Hnf4α^+/+, and Hnf4γ^+/+) or mice given vehicle were used as controls. Crypt and villus cells were isolated, incubated with fluorescently labeled fatty acids or glucose analog, and analyzed by confocal microscopy. Fatty acid oxidation activity and tricarboxylic acid (TCA) cycle metabolites were measured in cells collected from the proximal half of the small intestine of Hnf4αγ^DKO and control mice. We performed chromatin immunoprecipitation and gene expression profiling analyses to identify genes regulated by HNF4 factors. We established organoids from duodenal crypts, incubated them with labeled palmitate or acetate, and measured production of TCA cycle metabolites or fatty acids. Acetate, a precursor of acetyl coenzyme A (CoA) (a product of fatty acid β-oxidation [FAO]), or dichloroacetate, a compound that promotes pyruvate oxidation and generation of mitochondrial acetyl-CoA, were used for metabolic intervention.

Results: Crypt cells rapidly absorbed labeled fatty acids, and messenger RNA levels of Lgr5⁺ stem cell markers (Lgr5, Olfm4, Smoc2, Msi1, and Ascl2) were down-regulated in organoids incubated with etomoxir, an inhibitor of FAO, indicating that FAO was required for renewal of ISCs. HNF4A and HNF4G were expressed in ISCs and throughout the intestinal epithelium. Single knockout of either HNF4A or HNF4G did not affect maintenance of ISCs, but double-knockout of HNF4A and HNF4G resulted in ISC loss; stem cells failed to renew. FAO supports ISC renewal, and HNF4 transcription factors directly activate FAO genes, including Acsl5 and Acsf2 (encode regulators of acyl-CoA synthesis), Slc27a2 (encodes a fatty acid transporter), Fabp2 (encodes fatty acid binding protein), and Hadh (encodes hydroxyacyl-CoA dehydrogenase). In the intestinal epithelium of Hnf4αγ^DKO mice, expression levels of FAO genes, FAO activity, and metabolites of TCA cycle were all significantly decreased, but fatty acid synthesis transcripts were increased, compared with control mice. The contribution of labeled palmitate or acetate to the TCA cycle was reduced in organoids derived from Hnf4αγ^DKO mice, compared with control mice. Incubation of organoids derived from double-knockout mice with acetate or dichloroacetate restored stem cells.

Conclusions: In mice, the transcription factors HNF4A and HNF4G regulate the expression of genes required for FAO and are required for renewal of ISCs.

Keywords: Gene Expression; Mitochondria; Regulatory Networks; Stemness.

Figure 1.. FAO is required for intestinal stem cell renewal.

(A-E) Intestinal crypts uptake fatty acid more readily than villi (n=3 independent experiments). Crypt and villus epithelial cells were isolated from the proximal half of the small intestine (from the midpoint between gastroduodenal junction to ileal-cecal junction) and incubated with 10 μM BODIPY™ FL C16 (fluorescent palmitic acid), FL C12 (fluorescent dodecanoic acid), or 10 μM 2-NBDG (fluorescent glucose analog), respectively. Similar results are seen in duodenum, jejunum and ileum (Fig. S1B). (A) Time-course study of fluorescent fatty acid (BODIPY™ FL C16) uptake shows that intestinal crypt cells uptake more fatty acid than villus cells (scale bar, 50 μm). (B-C) Fatty acid (green fluorescence) accumulates more in the crypt cells than in villus cells (scale bars, 50 μm). (D) Red fluorescent fatty acids (10 μM BODIPY™ FL C12, 5 min) are observed in Lgr5-GFP stem cells in the crypts (scale bar, 5 μm). (E) Glucose uptake in crypt and villus cells (scale bar, 50 μm). (F) Fewer Lgr5-GFP cells are observed in organoids treated with Etomoxir (fatty acid oxidation inhibitor) for 72 hours (n=3 independent experiments). (G) qRT-PCR shows a significant decrease in the transcript levels of intestinal Lgr5⁺ stem cell markers in the organoids treated with 100 μM Etomoxir for 72 hours. Data are presented as mean ± SEM (n=3–4 independent organoid cultures per treatment, Student’s t-test, two-sided at P < 0.001***).

Figure 2.. HNF4 paralogs bind to and activate FAO genes, and FAO is compromised upon HNF4 loss.

(A-C) GSEA of RNA-seq (duodenal epithelium, 2–3 days post tamoxifen-induced knockout, n=3 biological replicates, Kolmogorov-Smirnov test) reveals significantly reduced expression of genes related to lipid digestion and fatty acid β-oxidation in Hnf4αɣ^DKO. Heatmaps of RNA-seq data show (D-E) decreased transcript levels of mitochondrial and peroxisomal β-oxidation genes but (F) increased transcript levels of lipogenesis genes in Hnf4αɣ^DKO (n=3 biological replicates, FDR < 0.05). Asterisks indicate genes that are directly bound by HNF4 within 50kb of annotated TSSs by ChIP-seq. (G) ChIP-seq (n=2 biological replicates) and RNA-seq (n=3 biological replicates) tracks show that HNF4 factors bind to and activate Acsl5 (acyl-CoA synthetase gene), and loss of HNF4 results in reduced active chromatin signal (H3K27ac ChIP-seq, n=2 biological replicates, see black dashed rectangles). (H) HNF4 factors can also bind to and activate Abcd1 (peroxisomal β-oxidation related transporter). The neighboring gene locus is not bound by HNF4 and shows no change in expression levels, serving as an internal control (Bcap31, see gray dashed rectangles). More examples are shown in Fig. S2. (I) Hnf4αɣ^DKO mice show decreased FAO activity in both villus and crypt cells. The FAO assay measures palmitoyl-CoA-induced oxidation, as detected by NADH generation. Data are presented as mean ± SEM (villus, n=9 biological replicates; crypt, n= 3–5 biological replicates; Student’s t-test, two-sided at P < 0.05*). (J) TCA metabolites are reduced upon HNF4 loss. Metabolites were extracted from villus cells scraped from proximal small intestine of Hnf4αɣ^DKO and their WT littermates after 3 consecutive days of tamoxifen or vehicle injection, respectively. Corrected ion counts were normalized by tissue weight, and further normalized relative to the average abundance of metabolite from WT littermates of the same experimental batch. Data are presented as mean ± SEM (LC-MS, n=10 WT and 9 mutants, Student’s t-test, two-sided at P < 0.01** and P < 0.05*).

Figure 3.. Loss of HNF4 paralogs in the intestinal epithelium triggers Lgr5⁺ stem cell loss.

(A) ATAC-seq of isolated intestinal stem cells reveals that HNF4A and HNF4G DNA-binding motifs (HOMER de novo) are most abundant at accessible chromatin regions of intestinal stem cells (GSE83394, n=2 biological replicates). (B) RNA-seq tracks of Hnf4α and Hnf4ɣ transcript levels in the intestinal stem cells (GSE83394, n=3 biological replicates) show robust expression of these paralogs. (C) Immunostaining of HNF4A and HNF4G in WT and Hnf4αɣ^DKO (representative of 3 biological replicates). (D) Immunostaining of OLFM4 (stem cell marker). Stem cells (red arrows) are lost in the crypts following Hnf4α and Hnf4ɣ double knockout. (E) qRT-PCR shows a significant decrease in the Lgr5⁺ stem cell markers in Hnf4αɣ^DKO. Crypts were isolated from duodenal epithelium of Hnf4αɣ^DKO and their WT littermates after 2 consecutive days of tamoxifen or vehicle injection, respectively. qPCR data are presented as mean ± SEM (n=3 biological replicates, Student’s t-test, two-sided at P < 0.01** and P < 0.05*). (F) Lgr5-GFP⁺ cells are observed in the crypt bottom of control mice, but are completely lost in Hnf4αɣ^DKO after 4 days of tamoxifen-induced knockout (representative of 3 biological replicates). (G) Immunostaining of HNF4A shows that Cre-mediated recombination of the Hnf4α locus is initiated via Lgr5-EGFP-Cre^ERT2 ( a stem cell-specific Cre driver).However, most recombined HNF4A-negative cells are not preserved in the epithelium of the Hnf4αɣ^DKO mice (representative of 3 biological replicates), suggesting that Hnf4αɣ^DKO mutant stem cells are replaced by neighboring cells. (H) Immunostaining of cleaved Caspase-3 in WT and Hnf4αɣ^DKO. (I) Hnf4αɣ^DKO shows a significant increase of cleaved Caspase-3 postive cells in the crypt base of Hnf4αɣ^DKO compared to WT littermates. Data are presented as mean ± SEM (n=3–4 biological replicates, Student’s t-test, two-sided at P < 0.05*).

Figure 4.. Transit amplifying-like proliferating cells predominate in the absence of Lgr5⁺ cells upon HNF4 loss.

(A) WT organoids exhibit branched structures and express robust Lgr5-GFP, whereas Hnf4αɣ^DKO organoids form spherical structures and exhibit little Lgr5-GFP fluorescence (see arrows). 1 μM tamoxifen or vehicle control was added to culture medium of primary organoids on Day 2 after seeding. After passage, Hnf4αɣ^DKO organoids failed to survive (n=6 independent experiments). Scale bar, 50 μm. Tamoxifen-induced Cre-only controls were also tested and shown in Fig. S4F. (B) WT and Hnf4αɣ^DKO organoids were passaged three times, and the number of surviving cells of organoids (n=4 independent organoid cultures) was counted at each passage, from primary (P0) passage to the third passage (P3). While HNF4 double-mutant organoids grow initially as proliferative spheres, they cannot be passaged indefinetly, such as their control counterparts. (C) Co-staining of EdU and Ki67 (proliferative markers). Organoids were treated with 10 μM EdU 6 hours prior to fixation. Scale bar, 50 μm. qRT-PCR shows a significant decrease of Lgr5⁺ stem cell markers (D) but a significant increase of proliferative cell markers (E) in Hnf4αɣ^DKO organoids. 1 μM tamoxifen or vehicle control was added to culture medium of primary organoids on Day 2 after seeding. All the primary organoids were harvested at Day 6 after seeding. Data are presented as mean ± SEM (n=3 independent organoid cultures, Student’s t-test, two-sided at P < 0.001***). (F) Crypt elongation is observed in Hnf4αɣ^DKO at day 4 post tamoxifen-induced knockout as shown in Ki67 immunofluorescent staining (representative of 4 biological replicates). (G) RNA-seq data reveal an elevation of proliferative cell markers in Hnf4αɣ^DKO compared to their littermate controls (n=3 biological replicates). Statistical tests were embedded in Cuffdiff at FDR < 0.001***.

Figure 5.. Loss of HNF4 paralogs leads to compromised TCA cycle contribution and elevated fatty acid synthesis.

(A-B) Schematics of ¹³C metabolic labeling experiments. (C) 24 hours of labeling with U-¹³C₁₆ palmitate (0.5 mM) indicates reduced contribution of β-oxidation to the TCA cycle metabolites in Hnf4αɣ^DKO organoids (n=4 independent organoid cultures, LC-MS, see expanded panels in Fig. S5A). (D-E) 6 hours of U-¹³C₂ acetate (2.5 mM) labeling shows (D) reduced ¹³C labeled TCA metabolites and (E) elevated ¹³C labeled fatty acids upon HNF4 loss (n=4 independent organoid cultures). Data are presented as mean ± SEM (Student’s t-test, two-sided at P < 0.001***, P < 0.01** and P < 0.05*).

Figure 6.. Acetate supplementation restores TCA cycle metabolites and ISC renewal.

(A) Schematic of agonists/antagonists used in the fatty acid/pyruvate oxidation-TCA pathways. CPT1: Carnitine palmitoyltransferase I; PDH: Pyruvate dehydrogenase; PDK: Pyruvate dehydrogenase kinase. (B) Schematics of acetate/DCA rescue experiments. (C-F) In Hnf4αɣ^DKO, intestinal stem cell maintenance is prolonged with addition of acetate (a precursor of acetyl-CoA) or dichloroacetate (DCA, a promoter of pyruvate oxidation and generation of acetyl-CoA). (C) Representative morphology (n=4 independent experiments) and (D) percentage of spherical organoids arising from organoid culture of Hnf4αɣ^DKO with or without acetate or DCA treatment (n=4 independent organoid cultures, one-way ANOVA followed by Dunnett’s post test at P < 0.001***). qRT-PCR shows rescued Lgr5⁺ stem cell markers in the Hnf4αɣ^DKO organoids upon (E) 25 mM acetate treatment (n=3 independent organoid cultures) or (F) 10 mM DCA treatment (n=4 independent organoid cultures). (G) TCA metabolite levels are restored in 25 mM acetate treated Hnf4αɣ^DKO organoids (n=4 independent organoid cultures). Scale bars, 50 μm.