J Biol Chem. 2013 Jun 14;288(24):17099-110.

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

Hayakawa K, Hirosawa M, Tabei Y, Arai D, Tanaka S, Murakami N, Yagi S, Shiota K.

Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Tokyo 113-8657, Japan.



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

PMID: 23625921



Here, we have demonstrated the multilayered epigenetic regulation by Sirt1, Ogt and Mgea5 in orexin neurogenesis.

This is the first report on the generation of orexin neurons from mouse embryonic stem cells (mESCs). N-acetyl-D-mannosamine ManNAc, which is a precursor for sialic acid, exhibited novel effects on epigenetic processes, including DNA demethylation, histone acetylation, and O-GlcNAcylation in orexin neurogenesis from mESCs (Fig. 1). In mESCs and neural precursor cells, the promoter region of orexin gene (Hcrt gene) are hypermethylated and H3/H4 are hypoacetylated. Hypoacetylation is established by Sirt1. Ogt, Ezh2 and Sin3A are also co-localized with the silencing complex. Loss of O-GlcNAcylation is a pivotal step in the transformation from the silent state with H3/H4 hypoacetylation to the active state with hyperacetylation at the Hcrt gene. ManNAc treatment caused de-localization of Sirt1, Ogt, Ezh2 and Sin3A, and recruitment of Mgea5, which have Oga and HAT activities. In this active state, other HATs such as p300 and CBP are also involved. We find it intriguingly that the process of generation of orexin neurons, central regulators of the whole body metabolism, comprises of an epigenetic mechanism consisting of nutrient-sensing molecules.

We propose that induced orexin neurons will provide a valuable tool in development of regenerative medicine applications.

Acknowledgements: This study was supported by the Advanced research for medical products Mining Program of the National Institute of Biomedical Innovation (NIBIO), Japan.


Figure 1 Proposed model of the epigenetic state in orexin and non-orexin neurons

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