LETTERS https://doi.org/10.1038/s42255-020-0267-9 1 Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Hefei Institute of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, China. 2 Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Institute for Stem Cell and Regeneration, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, China. 3 Fudan University, Shanghai, China. 4 These authors contributed equally: Linpeng Li, Keshi Chen, Tianyu Wang, Yi Wu. ✉ e-mail: liu_xingguo@gibh.ac.cn Somatic cell reprogramming provides insight into basic principles of cell fate determination, which remain poorly understood. Here we show that the transcription factor Glis1 induces multi-level epigenetic and metabolic remodelling in stem cells that facilitates the induction of pluripotency. We find that Glis1 enables reprogramming of senescent cells into pluripotent cells and improves genome stability. During early phases of reprogramming, Glis1 directly binds to and opens chromatin at glycolytic genes, whereas it closes chromatin at somatic genes to upregulate glycolysis. Subsequently, higher glycolytic flux enhances cellular acetyl-CoA and lactate lev- els, thereby enhancing acetylation (H3K27Ac) and lactylation (H3K18la) at so-called ‘second-wave’ and pluripotency gene loci, opening them up to facilitate cellular reprogramming. Our work highlights Glis1 as a powerful reprogramming factor, and reveals an epigenome–metabolome–epigenome signal- ling cascade that involves the glycolysis-driven coordination of histone acetylation and lactylation in the context of cell fate determination. Induced pluripotent stem cells (iPSCs) can be generated from human and mouse cell types by ectopic expression of the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc 1,2 . iPSCs hold great prom- ise in regenerative medicine therapy and for providing a valuable source of patient-specific cells to study and treat human diseases 3 . Furthermore, iPSC technology offers a unique experimental system for basic cell biologists to investigate the principles of cell fate deter- mination during development and its dysregulation in diseases. iPSC processes have been extensively studied and divided into stages according to distinct events at different regulatory levels: epigenetic, transcriptional, metabolic and morphological. At the genomic level, two main transcriptional waves and a successive wave of hydroxymethylation at enhancers have been proposed 4,5 . Chromatin dynamics shifting from open to closed and closed to open were revealed using an assay for transposase-accessible chro- matin with sequencing (ATAC–seq) 6 . At the metabolic level, a switch converting from somatic oxidative phosphorylation (OXPHOS) to a glycolytic-flux-dependent state and enhanced lipogenesis have been observed 7–13 . At the cellular level, the mesenchymal-to-epithelium transition (MET) at the initial stage of reprogramming is induced by Yamanaka factors 14–16 . An intermediate extraembryonic endoderm (XEN)-like state mediates the process of chemical reprogramming. Which is different from the primitive streak-like state induced by transcription factor reprogramming 3,17,18 . However, multi-level routes during reprogramming remain unclear. Metabolites such as acetyl-CoA, α-ketoglutarate (α-KG), S-adenosyl methionine (SAM), NAD + and O-linked beta-N-acetylglucosamine (O-GlcNAc) play critical roles in cell fate determination by regulation of epigenetic modifications and gene expression 19–22 . More recently, lactate-derived lactylation of histone was identified as a new epigen- etic modification 23 , which stimulates gene transcription from chro- matin in the late phase of M1 macrophage polarization 23 . However, whether lactate and lactate-derived histone lactylation are involved in pluripotency regulation remains unclear. Known as the ‘fifth Yamanaka reprogramming factor’, Gli-like transcription factor 1 (Glis1), a replacement for the oncogene c-Myc, improves the yield of green fluorescent protein- (GFP) posi- tive colonies, and the reprogramming activity of Glis1 is not depen- dent on the cell type or method used, thus suggesting that Glis1 has great clinical potential 17,24 . However, the mechanisms of Glis1, except for its interaction with Oct4, Sox2 and Klf4 during repro- gramming, remain obscure 25 . Here, we report an epigenome–metabolome–epigenome cascade initiated by the maternal transcription factor Glis1 during somatic cell reprogramming, indicating a unique multi-level path to plu- ripotency. Glis1 binds to and closes somatic genes while binding glycolysis genes to open them. This activates glycolysis, without affecting OXPHOS, thus increasing lactate and acetyl-CoA levels, and results in an increase in histone lactylation and acetylation on promoters of ‘second-wave’ and pluripotency genes. Glis1 is known to greatly enhance somatic cell reprogramming 25 (Extended Data Fig. 1a,b), so we asked whether it might also enable reprogramming of senescent somatic cells. We performed Sox2, Klf4 and Oct4 (SKO)-mediated reprogramming of mouse embryonic fibroblasts (MEFs) at a late passage (P7) (Extended Data Fig. 1c–e). Glis1 facilitates induction of pluripotency via an epigenome–metabolome–epigenome signalling cascade Linpeng Li  1,2,4 , Keshi Chen  1,2,4 , Tianyu Wang  1,2,4 , Yi Wu  1,2,4 , Guangsuo Xing  1,2 , Mengqi Chen  1,2 , Zhihong Hao  1,2 , Cheng Zhang  3 , Jinye Zhang  3 , Bochao Ma  1,2 , Zihuang Liu  1,2 , Hao Yuan  1,2 , Zijian Liu  1,2 , Qi Long  1,2 , Yanshuang Zhou  1,2 , Juntao Qi  1,2 , Danyun Zhao  1,2 , Mi Gao  1,2 , Duanqing Pei 1,2 , Jinfu Nie 1,2 , Dan Ye 3 , Guangjin Pan  1,2 and Xingguo Liu  1,2 ✉ NATURE METABOLISM | www.nature.com/natmetab