Epigenetic switching by the metabolism-sensing factors in the generation of orexin neurons from mouse embryonic stem cells

Abstract

The orexin system plays a central role in the integration of sleep/wake and feeding behaviors in a broad spectrum of neural-metabolic physiology. Orexin-A and orexin-B are produced by the cleavage of prepro-orexin, which is encoded on the Hcrt gene. To date, methods for generating other peptide neurons could not induce orexin neurons from pluripotent stem cells. Considering that the metabolic status affects orexin expression, we supplemented the culture medium with a nutrient factor, ManNAc, and succeeded in generating functional orexin neurons from mouse ES cells. Because DNA methylation inhibitors and histone deacetylase inhibitors could induce Hcrt expression in mouse ES cells, the epigenetic mechanism may be involved in this orexin neurogenesis. DNA methylation analysis showed the presence of a tissue-dependent differentially methylated region (T-DMR) around the transcription start site of the Hcrt gene. In the orexin neurons induced by supplementation of ManNAc, the T-DMR of the Hcrt gene was hypomethylated in association with higher H3/H4 acetylation. Concomitantly, the histone acetyltransferases p300, CREB-binding protein (CBP), and Mgea5 (also called O-GlcNAcase) were localized to the T-DMR in the orexin neurons. In non-orexin-expressing cells, H3/H4 hypoacetylation and hyper-O-GlcNAc modification were observed at the T-DMRs occupied by O-GlcNAc transferase and Sirt1. Therefore, the results of the present study suggest that the glucose metabolite, ManNAc, induces switching from the inactive state by Ogt-Sirt1 to the active state by Mgea5, p300, and CBP at the Hcrt gene locus.

Keywords: DNA Methylation; Embryonic Stem Cell; Epigenetics; Histone Acetylase; Histone Acetylation; Mgea5; Neurogenesis; O-GlcNAcylation; Ogt; Sirt1.

FIGURE 1.

Induction of orexin neurons from mESCs by treatment with ManNAc. A, shown is gene expression of hypothalamic transcription factors and peptides in the hypothalamus, fetal brain, and neural cells induced from mESCs by using SDIA, SDIA+BMP4, or gfCDM/SEFBq methods. RT(−) indicates PCR results for Actb without reverse transcription. B, shown is expression of Hcrt mRNA in neural cells differentiated from mESCs in the culture with SIDA+BMP4 medium containing 1 mm GlcN, GlcNAc, ManNAc, or Neu5Ac for 10 days. The mRNA expression was examined by RT-PCR (left) and RT-qPCR (right). Data represent the mean ± S.E. of triplicates of cell culture. The value of Hcrt and Npy expression for lanes 5 and 1, respectively, were set to 1. Asterisks indicate significant difference from lane 1 (Student's t test; **, p < 0.01). C, shown is an immunofluorescence assay of Orexin-A and -B in differentiated cells from mESCs in SDIA+BMP4 medium. The cells were probed for Orexin-A (green, upper), Tubb3 (red, upper), Orexin-B (green, middle), Ncam (red, middle), Dynorphin-A (red, bottom), and DAPI (blue). Scale bars represent 100 μm. Orexin-A and orexin-B positive cells were 6.2 ± 1.3% (n = 90) and 7.1 ± 1.9% (n = 90), respectively, of total cells in SDIA+BMP4 conditions with ManNAc. D, left, shown is RT-PCR of Hcrt in neural differentiated mESCs cultured under the gfCDM/SFEBq conditions in the presence of 1 mm GlcNAc and ManNAc. Right, shown is an immunofluorescence assay of Orexin-B in neural differentiated mESCs cultured under the gfCDM/SFEBq conditions in the presence of 1 mm ManNAc. Cells were probed for Orexin-B (green), Ncam (red), and DAPI (blue) after 25 days of gfCDM/SFEBq culture. Arrowheads indicate orexin-positive neuron. Scale bars denote 100 μm. Orexin-B-positive cells were 5.8 ± 1.1% (n = 90) of total cells in gfCDM/SFEBq conditions with ManNAc. E, shown is gene expression of orexin neural marker genes in neural cells differentiated from mESCs in SDIA+BMP4 medium containing 1 mm GlcN, GlcNAc, ManNAc, or Neu5Ac for 10 days. F, shown are the effects of GlcNAc, ManNAc, or Neu5Ac on the Hcrt mRNA expression during the differentiation period (early (days 0–4 and 0–70), late (4–10 and 7–10), or full (day 0–10)) in the SIDA+BMP4 medium. Expression of Hcrt was examined by RT-PCR. G, shown is a RT-PCR of Hcrt in differentiated neurospheres treated with 1 mm GlcNAc, ManNAc, and Neu5Ac. Each monosaccharide was added from the initiation of differentiation culture. H, shown is the response of Orexin-A secretion to high KCl levels in neural cells induced from mESCs by using SDIA+BMP4 medium supplemented with or without ManNAc. Cells were exposed to high KCl levels for 10 min, and Orexin-A in the medium was measured by conducting an ELISA assay. Data represent the mean ± S.E. of triplicates of cell culture. Asterisks indicate significant difference (Student's t test; **, p < 0.01). N.D., not determined. I, shown is dose-response of Orexin-A secretion to the ligands (ghrelin, leptin, and TRH) in neural cells differentiated from mESCs in SDIA+BMP4 medium. Orexin-A concentrations in the medium were measured by using ELISA.

FIGURE 2.

Epigenetic status of T-DMRs of the Hcrt gene by DNA methylation and histone modifications. A, shown is expression of Hcrt in mESCs after treatment with inhibitors of DNA methylation and histone deacetylation. mESCs were cultured for 48 h with 5 μm 5-aza-2′-deoxycytidine (5Aza), 200 nm trichostatin A (TSA), or 100 μm Zebularine (Zeb). B, shown is DNA methylation status of T-DMRs of Hcrt gene around the transcriptional start site. Top, shown is a schematic diagram of genes. The vertical lines denote the positions of cytosine residues of CpG sites. The thick horizontal line indicates the region of the ChIP-PCR fragment. Bottom, open and filled squares represent unmethylated and methylated cytosines, respectively. ORE1 indicates orexin regulatory element 1. Neural cells were differentiated from mESCs in SDIA+BMP4 medium. Orexin-A-positive (OrxA(+)) or Orexin-A-negative (OrxA(−)) colony was picked up by pipette after staining with an anti-Orexin-A antibody and then subjected to the bisulfite reaction. The red and green letters indicate the level of DNA methylation (%) at T-DMR-U (red square) and T-DMR-D (green square), respectively. C, shown are histone modifications and accumulation of epigenetic regulators at T-DMRs of the Hcrt gene locus. A ChIP assay was performed to determine the histone acetylation and methylation status and the accumulation of histone modification enzymes at regions 1 and 2 (upper diagram of Fig. 2B) in the T-DMR in differentiated neural cells from mESCs by culturing in SDIA+BMP4 medium in the presence or absence of 1 mm GlcNAc, ManNAc, or Neu5Ac. Input (10%) and normal IgG of rabbit and mouse (rIgG and mIgG) were used as positive and negative control, respectively. D, shown are nuclear and cytoplasmic levels of histone modification enzymes. E, shown is histone acetylation and methylation. Levels of histone modification enzymes and the histone modification status were detected by Western blotting of nuclear and cytoplasmic extracts prepared from neural differentiated mESCs cultured under SDIA+BMP4 conditions and treated with 1 mm GlcNAc, ManNAc, and Neu5Ac. Graphs indicate protein levels of Mgea5 and Sirt1 in nuclear and cytoplasmic fractions. The levels were estimated by the intensity of each band. Data represent the mean ± S.E. of three independent experiments. Asterisks indicate significant difference from lane 1 (Student's t test; **, p < 0.01; *, p < 0.05). F, shown is an immunofluorescence assay of Orexin-A, Mgea5, and Sirt1 in neural cells induced from mESCs by using SDIA+BMP4 medium supplemented with or without ManNAc. Scale bars denote 10 μm.

FIGURE 3.

Sirt1 contributes to the inactive state of the Hcrt gene. A, shown is expression of Hcrt mRNA in Sirt KO mESCs (Sirt^−/−) and in mESCs in the presence of a Sirt1 inhibitor (EX-527). RT-PCR (upper) and RT-qPCR (bottom) revealed expression of the Hcrt mRNA in undifferentiated, neural differentiated Sirt1^−/−mESCs and neural cells differentiated from mESCs in SDIA+BMP4 with or without ManNAc in the presence or absence of EX-527. WT indicates wild type mESCs. ManNAc (1 mm) and EX-527 (50 nm) were added to the medium on days 0 and 7, respectively. DMSO was a control of EX-527 treatment. RT-qPCR data represent the results of three independent experiments. Data represent the mean ± S.E. of three independent experiments. Asterisks indicate significant difference from lane 2 (Student's t test; **, p < 0.01). The value of Hcrt expression for lane 2 was set to 1. RT(−) indicates PCR results for Actb without reverse transcript. B, shown is an immunofluorescence assay of Orexin-A and -B in neural differentiated Sirt1^−/− mESCs cultured under SDIA+BMP4 conditions. Scale bars represent 100 μm. C, shown is the histone acetylation status of Hcrt T-DMRs by ChIP assay in neural differentiated Sirt1^−/− mESCs and EX-527-treated neural differentiated mESCs cultured under SDIA+BMP4 conditions in the presence or absence of ManNAc.

FIGURE 4.

O-GlcNAcylation system has a role of repressing Hcrt gene expression. A, shown is the accumulation of Ogt and O-GlcNAcylation at T-DMRs of Hcrt gene loci. ChIP assays of O-GlcNAcylation were performed using an antibody RL2. Rabbit IgG and RL2 absorbed with GlcNAc (200 mm) (Abs.) were negative controls, respectively. B, shown is the effect of an Ogt inhibitor and Oga inhibitor on Hcrt gene expression induced by ManNAc. RT-PCR and RT-qPCR of Hcrt in neural differentiated mESCs cultured under SDIA+BMP4 conditions containing an inhibitor of Ogt (BADGP) or Oga (Thiamet-G) are shown. BADGP or Thiamet-G was added to the medium from day 7. ManNAc (1 mm) was added from day 0. Data represent the mean ± S.E. of three independent experiments. Asterisks indicate significant difference from lane 1 (Student's t test; **, p < 0.01; *, p < 0.05). The value of Hcrt expression for lane 1 was set to 1. RT(−) indicates PCR results for Actb without reverse transcript. C, shown are the effects of overexpression of Ogt and Mgea5 on Hcrt gene expression. RT-qPCR of Hcrt in 3×FLAG-fused Ogt- and Mgea5-overexpressing neural differentiated mESCs cultured under the SDIA+BMP4 conditions in the presence of 1 mm ManNAc is shown. A vector, which expresses only 3×FLAG mRNA, was used as the control in the overexpression experiment. On day 7, cells were transfected with the overexpression vectors by using lipofection. At 10 days of culture, cells were collected, and RNA was isolated. Data represent the results of three independent experiments. Asterisks indicate significant difference from lane 1 (D) O-GlcNAcylation at the T-DMR in Ogt- or Mgea5-expressing cells. A ChIP assay of O-GlcNAcylation by RL2 in 3×FLAG-fused Ogt- and Mgea5-overexpressing neural differentiated mESCs cultured under the SDIA+BMP4 conditions in the presence of 1 mm ManNAc is shown. E, shown is co-accumulation of Ogt, Sirt1, Sin3A, and Ezh2 at T-DMR with the O-GlcNAcylation signal in the non-Hcrt expressing state. Interaction of O-GlcNAcylation was revealed by the Re-ChIP assay. RL2 or anti-Ogt was used as the first antibody. IP, immunoprecipitation.

FIGURE 5.

Dual function of Mgea5 as histone acetyltransferase and O-GlcNAcase at Hcrt T-DMRs. A, shown is a ChIP assay of histone acetylation in neural differentiated mESCs cultured under SDIA+BMP4 conditions in the presence of 5 μm Thiamet-G. Abs. indicates that RL2 absorbed with 200 mm GlcNAc. B, structure of Mgea5 with O-GlcNAcase and HAT domains. The amino acid residue at position 175 (Asp → Ala) or 891 (Tyr → Phe) was mutated to achieve deficiency of O-GlcNAcase or HAT activity, respectively. C, shown is the acetylation status of Mgea5 target residues, H3K14 and H4K8, in 3×FLAG-fused Mgea5 (WT, D175A, and Y891F)-overexpressing neural differentiated mESCs cultured under SDIA+BMP4 conditions in the presence of 1 mm ManNAc. Histone acetylation levels were detected by Western blotting of nuclear fractions. D, shown is a ChIP assay of O-GlcNAcylation and histone acetylation of Mgea5 target residues in Mgea5 (WT, D175A, and Y891F)-overexpressing neural differentiated mESCs cultured under SDIA+BMP4 conditions in the presence of 1 mm ManNAc. E, shown is the expression level of Hcrt in Mgea5 (WT, D175A, and Y891F)-overexpressing neural cells differentiated from mESCs. Expression levels of Hcrt were measured by using RT-PCR (left) and RT-qPCR (right). Data represent the mean ± S.E. of three independent experiments. Asterisks indicate significant difference from lane 1 (Student's t test; **, p < 0.01; *, p < 0.05). The value of Hcrt expression for lane 1 was set to 1. RT(−) indicates PCR results for Actb without reverse transcript. F, shown is the proposed model of the epigenetic state in orexin and non-orexin neurons.