IMMUNOLOGY 2017 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. The lysine deacetylase Sirtuin 1 modulates the localization and function of the Notch1 receptor in regulatory T cells Nimi Marcel, 1,2 * Lakshmi R. Perumalsamy, 1 * † Sanjay K. Shukla, 1,2 * Apurva Sarin 1,3‡ The ability to tune cellular functions in response to nutrient availability has important consequences for im- mune homeostasis. The activity of the receptor Notch in regulatory T (T reg ) cells, which suppress the functions of effector T cells, is indispensable for T reg cell survival under conditions of diminished nutrient supply. Anti- apoptotic signaling induced by the Notch1 intracellular domain (NIC) originates from the cytoplasm and is spa- tially decoupled from the nuclear, largely transcriptional functions of NIC. We showed that Sirtuin 1 (Sirt1), which is an NAD + (nicotinamide adenine dinucleotide)–dependent lysine deacetylase that inhibits NIC-dependent gene transcription, stabilized NIC proximal to the plasma membrane to promote the survival and function of activated T reg cells. Sirt1 was required for NIC-dependent protection from apoptosis in cell lines but not for the activity of the anti-apoptotic protein Bcl-xL. In addition, a variant NIC protein in which four lysines were mutated to arginines (NIC4KR) retained anti-apoptotic activity, but was not regulated by Sirt1, and reconstituted the functions of non- nuclear NIC in Notch1-deficient T reg cells. Loss of Sirt1 compromised T reg cell survival, resulting in antigen-induced T cell proliferation and inflammation in two mouse models. Thus, the Sirt1-Notch interaction may constitute an important checkpoint that tunes noncanonical Notch1 signaling. INTRODUCTION Several lines of evidence suggest that posttranslational modifications of key signaling nodes influence the outcome of signaling networks (1–4). Sirtuin 1 (Sirt1), an NAD + (nicotinamide adenine dinucleotide)– dependent deacetylase, regulates signaling pathways by modifying target proteins. Sirt1 is highly conserved and catalyzes the deacetylation of his- tones and of multiple nonhistone proteins, including Notch (5–11). Sirt1 inhibits Notch-mediated transcription either by directly repressing Notch targets (7, 12, 13) or by targeting the Notch intracellular domain (NIC) for degradation (6, 14). The Notch pathway signals through evolutionarily conserved ele- ments to regulate diverse processes, including cell survival. The pathway is activated upon binding of one of five ligands (Jagged 1 and 2, and Delta-like 1, 2, and 4) to the receptor proteins Notch1 to Notch4, which stimulates successive proteolytic cleavage events that result in the release of NIC from the full-length receptor. In the canonical pathway, NIC localizes to the nucleus and interacts with its transcriptional cofactors RBPJ-k (recombination signal binding protein for immunoglobulin kappa J) and MAML1 (mastermind-like transcriptional coactivator 1) to promote the transcription of target genes. Signaling through Notch1 is obligatory for the acquisition of T cell fate, although inputs from the Notch pathway are modulated during T cell development (15, 16). Emerging evidence has revealed a requirement for Notch proteins in the effector functions of mature T cells (17–19). Studies have also high- lighted differences in the Notch dependency of natural regulatory T (nT reg ) cells and induced T reg (iT reg ) cells, with Notch activity being essential for the expression of the gene encoding Foxp3 (forkhead box P3), a master regulator of T reg cell identity in iT reg cells but not nT reg cells (20–24). In another difference from iT reg cells, activated nT reg cells generated by being stimulated through CD3 and CD28 in vitro are char- acterized by the nonnuclear localization of NIC (25). Sirtuin activity is stimulated in nutrient-limiting conditions (26–28). Building on previous work, which demonstrated a requirement for Notch1 activity in promoting T reg cell survival in response to nutri- ent withdrawal (25), we asked whether Sirt1 modulated NIC activity. We present evidence implicating the Sirt1-NIC signaling axis in the anti-apoptotic activity in T reg cells and other cell types. We showed that the nonnuclear NIC localization was dependent on the deacetylase activity of Sirt1. We mapped the target sites of Sirt1 with recombinant Notch1 proteins to recapitulate these interactions in cell lines. Finally, we explored the consequences of this interaction for T reg cell function. RESULTS Sirt1 controls NIC localization in T reg cells Activated mouse T reg cells, which were generated through the stimula- tion of freshly isolated CD4 + CD25 + Foxp3 + T reg cells with beads coated with antibodies against CD3 and CD28 (see Materials and Methods), survived withdrawal of the cytokine interleukin-2 (IL-2) for 6 hours in culture (Fig. 1A). Under these conditions, T reg cells showed increased abundance of Sirt1 (Fig. 1, B and C, and fig. S1, A and B), and treatment with the Sirt inhibitor CHIC-35 abrogated T reg cell survival (Fig. 1A). We then investigated potential interactions between Sirt1 and NIC. The unusual localization of NIC in plasma membrane–proximal complexes in T reg cells is critical for its anti-apoptotic activity (25, 29). We found that the localization of NIC, which was detected by total internal reflection (TIRF) microscopy, was disrupted in CHIC-35–treated T reg cells in culture, with near-complete loss of signal at the plasma mem- brane for both NIC and the protein Rictor (Fig. 1D and fig. S1C) and a concomitant increase in the amounts of NIC and Rictor in the nucleus (Fig. 1E and fig. S1D). However, the localization of phosphatidylinositol 3-kinase (PI3K), a membrane-proximal lipid kinase also associated with NIC, was unchanged by CHIC-35 (Fig. 1, F and G, and fig. S1, C and D), 1 National Centre for Biological Sciences, Bengaluru, Karnataka 560065, India. 2 Manipal University, Manipal, Karnataka, India. 3 Institute for Stem Cell Biology and Regenerative Medicine, Bengaluru, Karnataka 560065, India. *These authors contributed equally to this work. †Present address: Department of Biotechnology, Indian Institute of Technology, Chennai, TN, India. ‡Corresponding author. Email: sarina@ncbs.res.in SCIENCE SIGNALING | RESEARCH ARTICLE Marcel et al., Sci. Signal. 10, eaah4679 (2017) 4 April 2017 1 of 13 on April 4, 2017 http://stke.sciencemag.org/ Downloaded from