Kynurenine, 3-OH-kynurenine, and anthranilate are nutrient metabolites that alter H3K4 trimethylation and H2AS40 O-GlcNAcylation at hypothalamus-related loci

Abstract

Epigenetic mechanisms can establish and maintain mitotically stable patterns of gene expression while retaining the DNA sequence. These mechanisms can be affected by environmental factors such as nutrients. The importance of intracellular dosages of nutrient metabolites such as acetyl coenzyme A and S-adenosylmethionine, which are utilized as donors for post-translational modifications, is well-known in epigenetic regulation; however, the significance of indirect metabolites in epigenetic regulation is not clear. In this study, we screened for metabolites that function as epigenetic modulators. Because the expression of genes related to hypothalamic function is reportedly affected by nutritional conditions, we used a neural cell culture system and evaluated hypothalamic-linked loci. We supplemented the culture medium with 129 metabolites separately during induction of human-iPS-derived neural cells and used high-throughput ChIP-qPCR to determine the epigenetic status at 37 hypothalamus-linked loci. We found three metabolites (kynurenine, 3-OH-kynurenine, and anthranilate) from tryptophan pathways that increased H3K4 trimethylation and H2AS40 O-GlcNAcylation, resulting in upregulated gene expression at most loci, except those encoding pan-neural markers. Dietary supplementation of these three metabolites and the resulting epigenetic modification were important for stability in gene expression. In conclusion, our findings provide a better understanding of how nutrients play a role in epigenetic mechanisms.

Figure 1

Epigenetic profile of H3K4me3 and H2AS40Gc in neurons cultured with selected metabolites. (a) Culture protocol for adding metabolites to human induced pluripotent stem cell (hiPSC)-derived neural cells. (b,c) Principal component analysis on ChIP-qPCR to determine H3K4me3 (b) and H2AS40Gc (c) levels. Red and black circles indicate significantly altered metabolites and non-treatment (Non), respectively.

Figure 2

Epigenetic activation at specific loci after supplementation of kynurenine, 3-OH-kynurenine, and anthranilate. Levels of H3K4me3 (a) and H2AS40Gc (b) on hypothalamic-neural genes in cells treated with metabolites. Heatmaps show ChIP-qPCR data. Values were normalized using input data. Color scale bars indicate histone modification level of each gene in treated cells, relative to non-treated cells. Dose-dependency of H3K4me3 (c) and H2AS40Gc (d) levels after kynurenine, 3-OH-kynurenine, and anthranilate supplementation. Kyn, kynurenine. 3-OH-kyn, 3-OH-kynurenine. Ant, anthranilate.

Figure 3

Elevated expression of genes encoding hypothalamic neuropeptides after supplementation with kynurenine, 3-OH-kynurenine, and anthranilate. (a) Results from RT-qPCR determining mRNA levels of neural peptide-coding genes, normalized to ACTB expression and visualized as a heatmap. Color scale bars indicate individual gene expression relative to expression in non-treated cells. (b) Immunofluorescent (IF) assays for neural peptides: GHRH, CARTPT, NPY, AGRP, CRH, and TRH. TUBB3 is a pan-neural marker. Scale bars, 200 μm.

Figure 4

Kynurenine, 3-OH-kynurenine, and anthranilate specifically increase H3K4me3 and H2AS40Gc levels. (a) Schematic depicting the kynurenine pathway of tryptophan metabolism. (b) Effect of supplementing tryptophan and kynurenine-pathway metabolites on H3K4me3 and H2AS40Gc levels at hypothalamic neural peptide-coding loci.

Figure 5

Response of gene expression to supplementing cells with quinolinate and NAD+, both located downstream of kynurenine in the kynurenine pathway. Gene expression was measured in neurons at day 24 using RT-qPCR, then normalized to ACTB expression. Means ± SD (n = 3). Relative values were based on the expression of non-treated cells equaling 1. TUBB3 and MAP2 were used as pan-neural markers. *P < 0.05.

Figure 6

Epigenetic activation by kynurenine, 3-OH-kynurenine, and anthranilate is necessary for maintenance of expression of hypothalamic neural peptide-coding genes. (a) Culture protocol for analyzing gene expression in long-term-cultured cells. Kynurenine, 3-OH-kynurenine, anthranilate, and quinolinate (all 100 μM) were added on day 14. Culturing continued until day 24, followed by 9 days of culture without metabolites. (b) Neural peptide-coding gene expression, evaluated with RT-qPCR, in neurons cultured without metabolites for 9 days. Means ± SD (n = 3). TUBB3 and MAP2 were used as pan-neural markers.

Figure 7

Supplementation of kynurenine, 3-OH-kynurenine, and anthranilate did not alter expression of histone-modification-enzyme-coding genes or NAD concentration. (a) Expression of histone-modifier-encoding genes was evaluated using RT-qPCR, normalized to ACTB, and visualized as a heatmap. Color scale bars indicate relative expression to non-treated cells. (b) Concentrations of intracellular NAD+ and NADH in neurons supplemented with kynurenine, 3-OH-kynurenine, anthranilate, or NAD+. Means ± SD (n = 3). *P < 0.05.