In the past year, our efforts focus on the role of SIRT1 in intestinal tissue homeostasis, the role of SIRT1 in ES cell biology and animal development, as well as the role of a special protein acylation in regulation of cellular metabolism. SIRT1 is a key regulator of animal development. However, despite the fact that the developmental defects of the first SIRT1 KO mouse model was reported more than 10 years ago, the molecular mechanisms underlying this important function of SIRT1 remain largely unclear. Using embryonic stem cells (ESCs) and mice as models, we recently discovered that SIRT1 contributes to the maintenance of homeostatic retinoic acid (RA) signaling and modulates mouse ESC differentiation. We further found that SIRT1 deficiency induced developmental defects is associated with elevated RA signaling in mice (Tang et al., Molecular Cell, 2014). SIRT1 is extremely highly expressed in preimplantation embryos and pluripotent mESCs derived from the inner cell mass of a blastocyst compared to differentiated cells. During the course of above study, we found that deletion or knockdown of SIRT1 in pluripotent mESCs reduces their maintenance, suggest that SIRT1 has a direct role in maintaining the pluripotent stem cell state of mESCs in addition to their differentiation. Given that SIRT1 is a well-established cellular metabolic sensor and regulator, we investigated whether metabolic dysregulation plays a role in SIRT1 deficiency-induced compromise of mESC maintenance. Through a large scale unbiased metabolomic analysis, we discovered that SIRT1 deficiency in mESCs primarily impairs cellular methionine metabolism, particularly the conversion of methionine to S-adenosylmethionine (SAM). As a result, SIRT1 deficient mESCs had a reduced cellular SAM abundance and decreased histone methylation levels, resulting in a dramatic alteration of global gene expression profiles and a hypersensitivity to methionine depletion/restriction-induced differentiation and apoptosis. Mechanistically we showed that SIRT1 promotes SAM production in part through Myc-mediated expression of methionine adenosyltransferase 2 (Mat2), an enzyme converting methionine to SAM in mESCs. Deletion of SIRT1 led to hyperacetylation of both N- and c-Myc proteins, two closely related key transcriptional regulators in ESCs, resulting in instability of c-Myc and reduced recruitment of these factors to the promoter of Mat2 and thereby reduced expression of this enzyme. Importantly, SIRT1 KO embryos had reduced Mat2a expression and histone methylation, and were sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increased the survival of SIRT1 KO newborn mice. Therefore, the defective methionine metabolism is partially responsible for SIRT1 deficiency-induced developmental defects in mice. Our findings uncover a novel regulatory mechanism for methionine metabolism, and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development. A paper for this study was published in 2017 in The EMBO Journal (Tang et al., EMBO Journal, 2017), and was selected as an Intramural Paper of the Month in November 2017. This study has also been highlighted recently in the Scientist Magazine (www.the-scientist.com/?articles.view/articleNo/51156/title/High-Throughput-Epigenetics-Analyses/). In the past decade, a panel of novel short-chain and long-chain lysine acylations (or lipid lysine acylations) has been identified in addition to protein acetylation. These lipid lysine acylations are associated with a number of cellular functions and dysregulation of these modifications have been associated with diseases. Although it has been shown that the classically annotated HDACs, including sirtuins, have deacylation activities beyond deacetylation, characterization of acyltransferases that mediate these modifications lags far behind that of deacylation enzymes. Moreover, our knowledge of the substrates and binding proteins of these modifications are still limited, and biological functions of these modification remain ambiguous. In the past two years, we have been focused on the regulation and function of Lysine 2-hydroxyisobutyrylation (Khib), a newly discovered lysine acylation. Khib is a widespread histone mark like lysine acetylation (Kac). However, molecular machineries mediating 2-hydroxyisobutyrylation remain elusive, and its substrate landscape and biological consequences are almost completely unknown. In one of our studies, through quantitative proteomics, global metabolomics, and functional metabolic analysis, we identified p300 as an acyltransferase for Khib, compared the p300-mediated Khib and Kac proteomics, and discovered a novel function of p300-mediated Khib in regulation of glycolysis. We discovered that p300 differentially regulates the Khib and Kac on distinct lysine sites, with only 6 out of the 149 p300-targeted Khib sites overlapping with the 693 p300-targeted Kac sites. We demonstrated that diverse cellular proteins, particularly glycolytic enzymes, are targeted by p300 for Khib but not for Kac. Specifically, deletion of p300 significantly reduces Khib levels on several p300-dependent, Khib-specific sites on key glycolytic enzymes including ENO1, decreasing their catalytic activities. Consequently, p300 deficient cells have impaired glycolysis and are hypersensitive to glucose depletion-induced cell death. Our study reveals a p300-catalyzed, Khib-specific molecular mechanism that regulates cellular glucose metabolism, and further indicate that p300 has an intrinsic ability to select short-chain acyl-CoA-dependent protein substrates. A paper describing this study was published in 2018 in Molecular Cell (Huag et al., Molecular Cell, 2018).

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12
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2018
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U.S. National Inst of Environ Hlth Scis
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Huang, He; Tang, Shuang; Ji, Ming et al. (2018) EP300-Mediated Lysine 2-Hydroxyisobutyrylation Regulates Glycolysis. Mol Cell 70:663-678.e6
Wang, Yongbin; Gan, Guifang; Wang, Bocheng et al. (2017) Cancer-associated Fibroblasts Promote Irradiated Cancer Cell Recovery Through Autophagy. EBioMedicine 17:45-56
Tang, Shuang; Fang, Yi; Huang, Gang et al. (2017) Methionine metabolism is essential for SIRT1-regulated mouse embryonic stem cell maintenance and embryonic development. EMBO J 36:3175-3193
Kwon, Sanghoon; Seok, Sunmi; Yau, Peter et al. (2017) Obesity and aging diminish sirtuin 1 (SIRT1)-mediated deacetylation of SIRT3, leading to hyperacetylation and decreased activity and stability of SIRT3. J Biol Chem 292:17312-17323
Choi, Sung E; Kwon, Sanghoon; Seok, Sunmi et al. (2017) Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver. Mol Cell Biol 37:
Ren, Natalie S X; Ji, Ming; Tokar, Erik J et al. (2017) Haploinsufficiency of SIRT1 Enhances Glutamine Metabolism and Promotes Cancer Development. Curr Biol 27:483-494
Wellman, Alicia S; Metukuri, Mallikarjuna R; Kazgan, Nevzat et al. (2017) Intestinal Epithelial Sirtuin 1 Regulates Intestinal Inflammation During Aging in Mice by Altering the Intestinal Microbiota. Gastroenterology 153:772-786
Lu, Jing; Xu, Qing; Ji, Ming et al. (2017) The phosphorylation status of T522 modulates tissue-specific functions of SIRT1 in energy metabolism in mice. EMBO Rep 18:841-857
Kazgan, Nevzat; Metukuri, Mallikarjuna R; Purushotham, Aparna et al. (2014) Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1?-FXR signaling and alters systemic bile acid homeostasis. Gastroenterology 146:1006-16
Yin, Huquan; Hu, Ming; Liang, Xiaomei et al. (2014) Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver. Gastroenterology 146:801-11

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