The EMBO Journal (2011) 30, 3242–3258 | & 2011 European Molecular Biology Organization | Some Rights Reserved 0261-4189/11 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL

Regulation of TFEB and V-ATPases by mTORC1 EMBO open

Samuel Pen˜ a-Llopis1,2,3,6, Eng, 2008). Mammalian TORC1 (mTORC1) includes the Silvia Vega-Rubin-de-Celis1,2,3,6, atypical serine/threonine kinase mTOR and an adaptor Jacob C Schwartz4, Nicholas C Wolff1,2,3, protein, regulatory-associated protein of mTOR (Raptor) Tram Anh T Tran1,2,3, Lihua Zou5, (Hara et al, 2002; Kim et al, 2002; Loewith et al, 2002). Xian-Jin Xie3, David R Corey4 and mTORC1 regulates cell growth, at least in part, by promoting James Brugarolas1,2,3,* cap-dependent translation and its best-characterized sub- strates are the eukaryotic initiation factor 4E binding 1 Department of Developmental Biology, University of Texas protein-1 (4E-BP1) (Sonenberg and Hinnebusch, 2009) and Southwestern Medical Center, Dallas, TX, USA, 2Department of Internal Medicine, Oncology Division, University of Texas Southwestern Medical S6 kinase 1 (S6K1) (Fingar and Blenis, 2004). mTORC1 is Center, Dallas, TX, USA, 3Simmons Comprehensive Cancer Center, specifically inhibited by rapamycin (also called sirolimus) University of Texas Southwestern Medical Center, Dallas, TX, USA, (Jacinto et al, 2004; Sarbassov et al, 2004) and rapamycin 4 Department of Pharmacology, University of Texas Southwestern analogues are used clinically for many applications including Medical Center, Dallas, TX, USA and 5Cancer Program, Broad Institute cancer treatment (Abraham and Eng, 2008). Indeed, two of Harvard and MIT, Cambridge, MA, USA rapamycin analogues, temsirolimus (Hudes et al, 2007) and Mammalian target of rapamycin (mTOR) complex 1 everolimus (Motzer et al, 2008), are approved by the FDA for (mTORC1) is an important, highly conserved, regulator the treatment of advanced RCC (renal cell carcinoma). of cell growth. Ancient among the signals that regulate mTORC1 is regulated by a complex formed by the proteins mTORC1 are nutrients. Amino acids direct mTORC1 to the tuberous sclerosis complex 1 (TSC1) and 2 (TSC2), which is surface of the late endosome/lysosome, where mTORC1 essential for the relay of signals from oxygen (Brugarolas becomes receptive to other inputs. However, the interplay et al, 2004; Connolly et al, 2006; Kaper et al, 2006; Liu et al, between endosomes and mTORC1 is poorly understood. 2006; DeYoung et al, 2008), energy stores (Inoki et al, 2003b; Here, we report the discovery of a network that links Corradetti et al, 2004; Shaw et al, 2004) and growth factors mTORC1 to a critical component of the late endosome/ (Jaeschke et al, 2002; Kwiatkowski et al, 2002; Zhang et al, lysosome, the V-ATPase. In an unbiased screen, we found 2003a). mTORC1 regulation by hypoxia involves the protein that mTORC1 regulated the expression of, among other regulated in development and DNA damage response 1 lysosomal genes, the V-ATPases. mTORC1 regulates (REDD1), a conserved protein with a novel fold (Vega- V-ATPase expression both in cells and in mice. V-ATPase Rubin-de-Celis et al, 2010), which, when overexpressed is regulation by mTORC1 involves a transcription factor sufficient to inhibit mTORC1 in a TSC1/TSC2-dependent translocated in renal cancer, TFEB. TFEB is required for manner (Brugarolas et al, 2004). Interestingly, in some the expression of a large subset of mTORC1 responsive settings, hypoxia signals are transduced via AMP-activated genes. mTORC1 coordinately regulates TFEB phosphoryla- protein kinase (Liu et al, 2006; Wolff et al, 2011), which tion and nuclear localization and in a manner dependent phosphorylates TSC2 and is normally involved in the relay of on both TFEB and V-ATPases, mTORC1 promotes endocy- energy signals (Inoki et al, 2003b; Gwinn et al, 2008). tosis. These data uncover a regulatory network linking an mTORC1 regulation by growth factors involves TSC2 phos- oncogenic transcription factor that is a master regulator of phorylation by Akt (Dan et al, 2002; Inoki et al, 2002; lysosomal biogenesis, TFEB, to mTORC1 and endocytosis. Manning et al, 2002; Potter et al, 2002), extracellular signal- The EMBO Journal (2011) 30, 3242–3258. doi:10.1038/ regulated kinase (Erk) (Ma et al, 2005) and ribosomal S6 emboj.2011.257; Published online 29 July 2011 kinase (Rsk) (Roux et al, 2004). TSC2 functions as a GTPase- Subject Categories: membranes & transport; signal transduction activating protein towards Ras homologue enriched in brain Keywords: autophagy; lysosome; microarray; RCC; Tcfeb (Rheb) (Castro et al, 2003; Garami et al, 2003; Tee et al, 2003; Inoki et al, 2003a; Zhang et al, 2003b), a small GTPase that directly interacts with and activates mTORC1 (Sancak et al, 2007; Avruch et al, 2009). Introduction mTORC1 regulation by nutrients is independent of TSC1/TSC2 (Zhang et al, 2003a; Smith et al, 2005; Roccio Target of rapamycin (TOR) complex 1 (TORC1) is a pivotal et al, 2006) and involves its localization to a cellular compart- regulator of cell growth conserved from yeast to humans and ment where it becomes receptive to activation by Rheb implicated in cancer (Wullschleger et al, 2006; Abraham and (Sancak et al, 2008, 2010). Amino-acid stimulation drives mTORC1 to the surface of the late endosome/lysosome in *Corresponding author. Department of Internal Medicine, Oncology a manner that depends on a Rag GTPase heterodimer (RagA Division, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, NB5.102A, Dallas, TX 75390-9133, USA. [or B] bound to RagC [or D]) and a multimeric complex Tel.: þ 1 214 648 4059; Fax: þ 1 214 648 1960; termed the Ragulator (Kim et al, 2008; Sancak et al, 2010). E-mail: [email protected] Importantly, constitutive mTORC1 targeting to this compart- 6 These authors contributed equally to this work ment renders mTORC1 insensitive to amino-acid withdrawal Received: 21 December 2010; accepted: 24 June 2011; published (Sancak et al, 2010). The significance of the late endosome/ online: 29 July 2011 lysosome in mTORC1 activation has been recently established

3242 The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al in experiments manipulating endosome maturation (Flinn (Figure 1C). Given these results and assuming a binomial et al, 2010; Li et al, 2010). Disrupting endosome maturation distribution with equal probabilities for all patterns, the with a constitutively active Rab5 or through depletion of probability of finding 78 probes with an LLHL pattern by hVps39 inhibited the activation of mTORC1 by growth factors chance alone was below 1.6 1028. and amino acids (Flinn et al, 2010; Li et al, 2010). To validate the microarray findings, qRT–PCR was per- A critical component of the endosome required for its formed on a subset of genes with the weakest LLHL associa- acidification and maturation is the vacuolar H þ -ATPase tion (highest P-values). In every instance, the LLHL pattern (V-ATPase) (Marshansky and Futai, 2008). V-ATPases are was confirmed and the fold change was even greater than multisubunit complexes formed by a membrane-embedded expected (Figure 1E). 0 00 V0 domain (a, d, e, c, c and c subunits) responsible for Among the LLHL probes, we found six for V-ATPase genes proton translocation, and a cytosolic V1 domain (A–H sub- (Figure 1D; Supplementary Table S1). The probability that units), which provides energy through ATP hydrolysis there would be six V-ATPase probes among the 78 probes (Forgac, 2007). V-ATPases are present in virtually every identified by chance alone was o9 108. Furthermore, an eukaryotic cell and V-ATPase function is essential for survival analysis of all the V-ATPase probes in the array showed under a variety of conditions (Beyenbach and Wieczorek, a striking LLHL pattern for most genes (Figure 2A) and the 2006). However, little is known about the regulation of results were confirmed in a subset of genes by qRT–PCR V-ATPases and in particular about how their expression is (Figure 2B) and western blot (Figure 2C). controlled. Because Tsc2 þ / þ and Tsc2/ cells are also p53/, they Here, we have uncovered a regulatory network linking may have diverged from each other over time, and thus, we V-ATPases and endocytosis to mTORC1 that involves the examined the role of mTORC1 in V-ATPase gene expression in transcription factor EB (TFEB). TFEB is a basic helix-loop-helix primary MEFs. MEFs were generated from Tsc1floxP/floxP (bHLH) leucine zipper transcription factor of the Myc family, (Tsc1F/F) (Kwiatkowski et al, 2002) as well as littermate microphthalmia transcription factor subfamily (Steingrimsson Tsc1 þ / þ embryos, and exposed to recombinant adenoviruses et al, 2004), that is translocated in a subset of renal tumours driving the expression of Cre recombinase (Ad-Cre). Ad-Cre (Davis et al, 2003; Kuiper et al, 2003; Srigley and Delahunt, transduction of Tsc1F/F MEFs led to effective recombination 2009). of loxP sites (Supplementary Figure S1A), with loss of the Tsc1 protein, activation of mTORC1 (Figure 2D) and V-ATPase upregulation (Figure 2D). Results To broaden these observations, we examined publicly V-ATPase enrichment among mTORC1-regulated genes available gene expression data sets. Recently, a study was We identified V-ATPase genes in an unbiased screen for genes reported in which the effects of rapamycin were evaluated whose expression was tightly regulated by mTORC1. We used over time in serum-deprived Tsc2/ and Tsc1/ immorta- a robust experimental paradigm involving the combination lized MEFs (Duvel et al, 2010). While in this study rapamycin of two interventions, one genetic and one pharmacologic. was administered only to the mutant cells, we observed a In Tsc2 þ / þ immortalized mouse embryo fibroblasts (MEFs), previously unrecognized downregulation of V-ATPase expres- but not Tsc2/, mTORC1 is inhibited by serum deprivation sion by rapamycin (Supplementary Figure S2). V-ATPase (0.1% fetal bovine serum, FBS) (Figure 1A). In Tsc2/ cells, expression was reduced by 6 h (Supplementary Figure S2) abnormally increased mTORC1 activity can be corrected by and maximal V-ATPase downregulation was observed treatment with rapamycin (Figure 1B). By contrast, rapamy- at 24 h, the time at which our experiments were conducted cin has little effect on mTORC1 in Tsc2 þ / þ cells in which (Figure 1D). Similar results were observed in a second mTORC1 is already inhibited by low serum (Figure 1B). Thus, publicly available data set of immortalized Tsc2/ MEFs under serum-deprived conditions, mTORC1 activity is low in treated with rapamycin (Cunningham et al, 2007) (Supple- Tsc 2 þ / þ cells (untreated or rapamycin treated), high in Tsc2/ mentary Figure S3). The analysis of these data sets confirmed cells, but lowered by rapamycin; a pattern referred to as a ‘low/ our results that mTORC1 regulated V-ATPase expression. low/high/low’ or ‘LLHL’ (Figure 1B). This experimental setup creates an ‘asymmetric’ response to rapamycin (in Tsc2/ mTORC1 regulation of V-ATPase expression in versus Tsc2 þ / þ cells), which, as illustrated hereafter, can be the mouse harnessed to great advantage. To extend our observations to an in vivo setting, we injected Using an Affymetrix MOE430A array platform and Tsc1F/F mice with Ad-Cre intravenously under conditions duplicate samples, genes were identified with an LLHL known to induce recombination of target genes in hepato- expression pattern (upregulated in untreated Tsc2/ cells cytes (Kucejova et al, 2011). Ad-Cre (but not control Ad-eGFP compared with a baseline formed by Tsc2 þ / þ cells, untreated or saline) led to effective recombination of loxP sites or rapamycin treated, and Tsc2/ rapamycin-treated cells). (Supplementary Figure S1B), loss of the Tsc1 protein and With a false discovery rate-corrected P-value (FDR Q) cutoff robust activation of mTORC1 (Figure 2E). This was accom- of 0.05 and a threshold of 1.5-fold, we identified 78 probe sets panied by increased expression of V-ATPase genes at both the (henceforth referred to as probes). They corresponded to 75 mRNA and protein levels (Figure 2E and F; Supplementary genes (Figure 1C and D). Figure S4A). As Ad-Cre injection affected the expression of We hypothesized that if the association of probes with the many housekeeping genes including actin, we used as a LLHL pattern was random, there would be similar numbers of reference cyclophilin B, whose expression was seemingly probes with the three alternate patterns (HLLL, LHLL and unaffected by Ad-Cre (Figure 2F; Supplementary Figure S4). LLLH). Strikingly, however, no probes fitted an HLLL or LHLL While control mice were infected also with an adenovirus pattern, and only 22 probes conformed to an LLLH pattern (Ad-GFP), we could not rule out effects of Cre recombinase

&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 16 | 2011 3243 Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

A Tsc2 +/+ Tsc2 –/– D Tsc2+/+ Tsc2 –/– % FBS 10 0.1 10 0.1 Symbol Veh Rapa Veh Rapa P FDR Q FC Gene title Fam50a 7E–7 0.005 1.5 Family with sequence similarity 50, member A Tsc2 Tuba4a 3E–6 0.010 1.9 Tubulin, α 4A Scpep1 9E–6 0.010 1.5 Serine carboxypeptidase 1 Sord 1E–5 0.010 2.9 Sorbitol dehydrogenase P -S6K1 (T389) Anpep 2E–5 0.012 1.9 Alanyl (membrane) aminopeptidase Pfkfb3 2E–5 0.013 1.6 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 Atp6v0b 2E–5 0.013 1.7 ATPase, H+ transporting, lysosomal V0 subunit B S6K1 Npy 3E–5 0.013 2.1 Neuropeptide Y Tuba4a 3E–5 0.013 2.3 Tubulin, α 4A Rpp25 4E–5 0.015 1.91 9 Ribonuclease P 25 subunit (human) P -S6 (S ) Mid1ip1 5E–5 0.015 1.6 Mid1 interacting protein 1 (gastrulation specific G12-like (zebrafish)) 235/236 Mrps6 7E–5 0.017 1.9 Mitochondrial ribosomal protein S6 Vps26a 7E–5 0.017 1.6 Vacuolar protein sorting 26 homologue A (yeast) S6 Flcn 8E–5 0.017 1.6 Folliculin Mt1 8E–5 0.017 1.6 Metallothionein 1 Cyc1 1E–4 0.018 1.5 Cytochrome c-1 Tubulin 2410022L05Rik 1E–4 0.018 1.6 RIKEN cDNA 2410022L05 gene Dclk1 1E–4 0.018 1.8 Doublecortin-like kinase 1 Snta1 1E–4 0.018 1.5 Syntrophin, acidic 1 Itm2a 1E–4 0.018 1.5 Integral membrane protein 2A Sesn3 1E–4 0.018 2.2 Sestrin 3 0.1% FBS Galc 1E–4 0.018 1.6 Galactosylceramidase B Neu1 2E–4 0.018 2.0 Neuraminidase 1 Tsc2 +/+ Tsc2 –/– Gga2 2E–4 0.018 1.5 Golgi-associated, γ adaptin ear containing, ARF-binding protein 2 Esd 2E–4 0.018 1.6 Esterase D/formylglutathione hydrolase Rapamycin –+ – + Chchd10 2E–4 0.018 1.9 Coiled-coil-helix-coiled-coil-helix domain containing 10 Hmox1 2E–4 0.019 2.3 Heme oxygenase (decycling) 1 Acot13 2E–4 0.020 1.6 Acyl-CoA thioesterase 13 P -S6K1 (T389) A030009H04Rik 3E–4 0.020 1.6 RIKEN cDNA A030009H04 gene Steap3 3E–4 0.020 1.6 STEAP family member 3 S6K1 Alad 3E–4 0.020 1.8 Aminolevulinate, Δ-, dehydratase Slc25a33 3E–4 0.020 1.6 Solute carrier family 25, member 33 Cndp2 3E–4 0.020 1.6 CNDP dipeptidase 2 (metallopeptidase M20 family) P -S6 (S ) Ube2l3 3E–4 0.022 1.5 Ubiquitin-conjugating enzyme E2L 3 235/236 Snapc3 4E–4 0.023 1.6 Small nuclear RNA activating complex, polypeptide 3 Kif1a 4E–4 0.023 1.5 Kinesin family member 1A Gats 4E–4 0.023 1.7 GATS protein-like 2 S6 Atp6v0b 5E–4 0.024 1.6 ATPase, H+ transporting, lysosomal V0 subunit B Renbp 5E–4 0.025 1.9 Renin-binding protein Il1rn 6E–4 0.026 1.5 Interleukin 1 receptor antagonist Tubulin Uchl3/Uchl4 6E–4 0.026 1.7 Ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase) and L4 Steap1 6E–4 0.026 2.3 Six transmembrane epithelial antigen of the prostate 1 L L H L Spon2 6E–4 0.027 1.8 Spondin 2, extracellular matrix protein Creg1 6E–4 0.027 2.9 Cellular repressor of E1A-stimulated genes 1 mTORC1 activity Nrk 7E–4 0.027 2.3 Nik-related kinase Lgals3 7E–4 0.027 4.3 Lectin, galactose binding, soluble 3 Hif1a 7E–4 0.027 1.6 Hypoxia-inducible factor 1, α subunit Maoa 7E–4 0.027 1.5 monoamine oxidase A Gla 7E–4 0.028 2.1 Galactosidase,  C No. of significant probesets (genes) Atp6v1g1 8E–4 0.028 1.6 ATPase, H+ transporting, lysosomal V1 subunit G1 Uap1l1 8E–4 0.028 2.2 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1 Cxadr 8E–4 0.029 1.8 Coxsackievirus and adenovirus receptor HLLL LHLL LLHL LLLH Abcb6 9E–4 0.029 1.7 ATP-binding cassette, subfamily B (MDR/TAP), member 6 Oxct1 9E–4 0.029 1.5 3-Oxoacid CoA transferase 1 FDR 0 0 537 (495) 41 (38) Ero1l 9E–4 0.029 2.1 ERO1-like (S. cerevisiae) Atp6ap2 9E–4 0.030 1.6 ATPase, H+ transporting, lysosomal accessory protein 2 FDR + 0 0 78 (75) 22 (20) 0610010O12Rik 0.001 0.031 1.6 RIKEN cDNA 0610010O12 gene FC>1.5 Pgp 0.001 0.034 1.6 Phosphoglycolate phosphatase P < 1.6×10–28 Lrpap1 0.001 0.034 1.5 Low-density lipoprotein receptor-related protein associated protein 1 Fmr1 0.002 0.035 1.6 Fragile X mental retardation syndrome 1 homologue Cyb5b 0.002 0.036 1.7 Cytochrome b5 type B Cdsn 0.002 0.039 1.6 Corneodesmosin E Emb 0.002 0.039 1.8 Embigin 7 Ddit3 0.002 0.040 1.6 DNA-damage-inducible transcript 3 *** Tsc2 +/+ Gpr137b 0.002 0.041 1.6 G protein-coupled receptor 137B 6 *** Tsc2 +/+ Rapa Sqstm1 0.003 0.043 1.5 Sequestosome 1 Bdh1 0.003 0.043 2.0 3-Hydroxybutyrate dehydrogenase, type 1 Tsc2 –/– 1110008P14Rik 0.003 0.044 1.6 RIKEN cDNA 1110008P14 gene 5 –/– -actin Tsc2 Rapa Slc16a1 0.003 0.045 2.4 Solute carrier family 16 (monocarboxylic acid transporters), member 1  4 Foxa2 0.003 0.046 1.6 Forkhead box A2 *** Cd68 0.003 0.047 2.7 CD68 antigen 3 *** Atp6v1a 0.003 0.047 1.5 ATPase, H+ transporting, lysosomal V1 subunit A *** Ddit4l 0.003 0.047 1.7 DNA-damage-inducible transcript 4-like 2 *** *** Steap1 0.003 0.047 1.6 Six transmembrane epithelial antigen of the prostate 1 Atp6v0c 0.004 0.048 1.5 ATPase, H+ transporting, lysosomal V0 subunit C 1 Fabp3 0.004 0.049 1.6 Fatty acid binding protein 3, muscle and heart Relative mRNA levels normalized to Vamp3 0.004 0.049 1.7 Vesicle-associated membrane protein 3 0 Dpp7 0.004 0.049 1.7 Dipeptidylpeptidase 7

–3 0 3 Ddit4l Fabp3 Dpp7 Atp6v1a Steap1 Atp6v0c Vamp3 Normalized expression Figure 1 Identification of mTORC1-regulated genes. Western blot of immortalized MEFs of the indicated genotypes grown for 24 h in the stated amounts of FBS (A), and in the presence of rapamycin (or vehicle) (B). (C) Number of probes (genes) in Affymetrix microarrays corresponding to the indicated patterns (upregulated under one condition compared with the other three by t-test using a Qo0.05 after FDR correction). Data are shown also incorporating a fold change criterion (41.5-fold; FC41.5). (D) Heatmap representation of normalized expression values for LLHL probes with a fold change 41.5 (ranked by P-value). Genes encoding V-ATPase subunits and V-ATPase-associated proteins are highlighted in yellow and in bold lysosomal genes are noted. (E) qRT–PCR analysis of genes with the weakest LLHL pattern. Error bars represent s.e.m. (n ¼ 4); ***Po0.001.

beyond loxP site recombination, and additional experiments Next, we explored the regulation of V-ATPases by mTORC1 were performed using Tsc1 þ / þ mice injected with Ad-Cre in a previously reported tumour model. Transgenic mice as a control (Figure 2F; Supplementary Figure S4B). expressing a constitutively active myristoylated form of Independently of the control, Tsc1 loss was accompanied by Akt1 (myr-Akt) in the prostate develop prostate intraepithe- mTORC1 activation and increased V-ATPase expression. lial neoplasia (PIN) and PIN lesions exhibit robust mTORC1

3244 The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

Tsc2 +/+ Tsc2 –/– +/+ A B 12 Tsc2 Symbol ––Rapa Rapa P FDR Q FC Gene title Tsc2 +/+ Rapa Atp6ap1 0.06 0.20 1.16 V-ATPase, accessory protein 1 10 *** Tsc2 –/– Atp6ap2 9E–4 0.030 1.62 V-ATPase, accessory protein 2 Tsc2 –/– Rapa

Atp6ap2 0.001 0.032 1.50 V-ATPase, accessory protein 2 -actin ***  8 Atp6ap2 0.009 0.07 1.49 V-ATPase, accessory protein 2 *** *** Atp6v0a1 0.016 0.10 1.55 V0-ATPase, subunit a1 6 Atp6v0a1 0.030 0.14 1.36 V0-ATPase, subunit a1 Atp6v0a1 0.16 1.25 V0-ATPase, subunit a1 *** 0.041 4 Atp6v0a1 0.96 0.98 1.01 V0-ATPase, subunit a1 *** *** Atp6v0a2 3E–4 0.021 1.25 V0-ATPase, subunit a2 Atp6v0a2 0.019 0.11 1.24 V0-ATPase, subunit a2 2 normalized to Atp6v0a2 0.78 0.88 –1.04 V0-ATPase, subunit a2 Relative mRNA levels Tcirg1 0.004 0.05 1.29 V0-ATPase, subunit a3 0 Atp6v0b 2E–5 0.013 1.66 V0-ATPase, subunit b v0c Atp6v0b 5E–4 0.024 1.59 V0-ATPase, subunit b Atp6v0b 0.008 0.07 1.27 V0-ATPase, subunit b Atp6ap2 Atp6v0b Atp6 Atp6v1a Atp6v1d Atp6v1b2 Atp6v1g1 Atp6v0c 0.004 0.048 1.50 V0-ATPase, subunit c Atp6v0c 0.07 0.23 1.15 V0-ATPase, subunit c Atp6v0c 0.10 0.27 1.11 V0-ATPase, subunit c +/+ –/– Atp6v0d1 0.008 0.07 1.24 V0-ATPase, subunit d1 Tsc2 Tsc2 Atp6v0e 0.44 0.64 1.07 V0-ATPase, subunit e C Atp6v0e2 0.57 0.74 –1.04 V0-ATPase, subunit e2 Rapamycin –+ – + Atp6v1a 0.003 0.047 1.50 V1-ATPase, subunit A Atp6v1a 0.007 0.07 1.75 V1-ATPase, subunit A Atp6v1a Atp6v1b2 4E–4 0.023 1.41 V1-ATPase, subunit B2 Atp6v1b2 7E–4 0.027 1.37 V1-ATPase, subunit B2 Atp6v1b2 Atp6v1c1 9E–4 0.029 1.46 V1-ATPase, subunit C1 Atp6v1c1 0.001 0.032 1.44 V1-ATPase, subunit C1 Tubulin Atp6v1c1 0.008 0.07 1.51 V1-ATPase, subunit C1 Atp6v1d 7E–5 0.017 1.45 V1-ATPase, subunit D Atp6v1d 0.002 0.036 1.33 V1-ATPase, subunit D Atp6v1d 0.002 0.039 1.35 V1-ATPase, subunit D D Vehicle Ad-Cre Atp6v1e1 0.001 0.034 1.42 V1-ATPase, subunit E1 +/+ F/F +/+ F/F Atp6v1e1 0.08 0.24 1.18 V1-ATPase, subunit E1 Tsc1 Tsc1 Tsc1 Tsc1 Atp6v1e2 0.78 0.88 –1.01 V1-ATPase, subunit E2 Rapamycin – + –+–+–+ Atp6v1f 0.08 0.24 1.25 V1-ATPase, subunit F Atp6v1g1 8E–4 0.028 1.62 V1-ATPase, subunit G1 Tsc1 Atp6v1g1 0.07 0.22 1.38 V1-ATPase, subunit G1 Atp6v1g2 0.036 0.15 1.09 V1-ATPase, subunit G2 Atp6v1h 0.004 0.05 1.50 V1-ATPase, subunit H Atp6v1a –3 0 3 Normalized expression Atp6v1b2 E Adenovirus Veh eGFP Cre P -S6K1

Days 10 3 3 2 2 3 3 5 5 7 7 10 10 (T389) Tsc1 P -S6 (S ) Tcirg1 235/236 (Atp6v0a3) S6 Atp6v1a Tubulin Atp6v1b2

P -S6K1 F (T ) 389 Tsc1+/+ 3.0 * * F/F S6K1 2.0 * Tsc1 2.5 P -S6 ** * 1.5 2.0 (S235/236) mRNA levels mRNA levels * 1.5 ** *** S6 1.0 1.0 P -4E-BP1 Atp6v1a 0.5 Atp6v1b2 (S65) 0.5 P -4E-BP1

normalized to cyclophilin 0.0 (T ) normalized to cyclophilin 0.0 37/46 0 7 14 21 35 0 7 14 21 35 Relative Relative Cyclophilin Ad-Cre treatment Ad-Cre treatment time (days) time (days) Figure 2 mTORC1 regulates V-ATPases. (A) Heatmap representation of normalized expression values for all informative V-ATPase probes in the arrays. qRT–PCR (B) and western blot (C) of V-ATPases in Tsc2 þ / þ and Tsc2/ MEFs in 0.1% FBS and treated (or not) with rapamycin for 24 h (n ¼ 4). (D) Western blot of Tsc1 þ / þ and Tsc1F/F primary MEFs exposed (or not) to Ad-Cre and treated (or not) with rapamycin in 0.1% FBS for 24 h. (E) Western blot from liver extracts (assembled from two gels) of mice injected with the indicated adenovirus (or vehicle) and harvested after the stated number of days. (F) qRT–PCR analysis of V-ATPase gene expression in liver extracts from Tsc1 þ / þ or Tsc1F/F mice injected with Ad-Cre and harvested following the indicated number of days (n ¼ 2 per time point). Error bars represent s.e.m.; *Po0.05; **Po0.01; ***Po0.001. activation (Majumder et al, 2004). Myr-Akt would be ex- While this represented a very different experimental para- pected to inactivate Tsc1/Tsc2 and the PIN phenotype is digm and gene expression studies were performed following reversed by treatment with the rapamycin analogue 12 and 48 h of RAD001 oral administration, a collective everolimus (also called RAD001) (Majumder et al, 2004). analysis showed a similar mTORC1-dependent induction of

&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 16 | 2011 3245 Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

V-ATPase expression (Supplementary Figure S5). In fact, a Among eight Tfeb shRNAs tested, two were found that statistically significant LLHL pattern was found for 13 probes substantially lowered Tfeb levels (Figure 4A and B). Tfeb corresponding to 9 V-ATPase genes. Thus, V-ATPases are depletion downregulated V-ATPase expression, particularly in regulated by mTORC1 not only in cells in culture, but also Tsc2-deficient cells, in which baseline levels were upregu- in mice in both physiological and pathological states. lated (Figure 4B; see also Figure 4E). These data show that Tfeb is required for mTORC1-induced V-ATPase expression. Transcriptional regulation of V-ATPases by mTORC1 To obtain insight into the mechanism of V-ATPase mRNA Tfeb is an important mediator of mTORC1 changes upregulation by mTORC1, we examined the relative contribu- on gene expression tion of mRNA synthesis and stability. While as expected, To determine how broadly TFEB regulated gene expression baseline V-ATPase mRNA levels were higher in Tsc2/ than downstream of mTORC1, we performed gene expression Tsc2 þ / þ cells, mRNA decay rates were very similar studies in MEFs stably depleted of Tfeb. Having previously (Figure 3A). These data suggested that V-ATPase mRNA used an Affymetrix platform and seeking to determine levels were upregulated by mTORC1 through a transcriptional the robustness of our findings, Illumina microarrays were mechanism. Consistent with this notion, RNA polymerase II used. Otherwise, the experimental design was identical as (Pol II) chromatin immunoprecipitation (ChIP) experiments that reported in Figure 1C and D. First, we compared the showed Pol II enrichment at V-ATPase coding sequences in results in the Tsc2 þ / þ and Tsc2/ cells transduced with a Tsc2/ cells (Supplementary Figure S6). This effect persisted scrambled shRNA to those previously obtained with plain after normalization to b-actin ChIP data suggesting that the Tsc2 þ / þ and Tsc2/ cells. While the number of LLHL effect was not due to a generalized increase in Pol II tran- probes identified with the Illumina chips was much larger scription by mTORC1 (Figure 3B). than with the Affymetrix arrays, the distribution across We noted that among the 75 genes regulated by mTORC1 patterns was strikingly similar; the majority of probes had was hypoxia-inducible factor 1a (Hif-1a) (Figure 1D), which an LLHL pattern and no probes were found with a HLLL or we previously showed is regulated by Tsc1/Tsc2 and mTORC1 LHLL pattern (compare Figures 4C and 1C). Furthermore, (Brugarolas et al, 2003). Hif-1a is a critical determinant of the among the 75 genes previously identified with a LLHL activity of the heterodimeric Hif-1 transcription factor pattern, a statistically significant LLHL pattern was retained (Kaelin, 2008) and a recent ChIP-chip study reported finding in 48% (Supplementary Figure S7). Considering that the Hif-1a on V-ATPase d1 sequences (Xia et al, 2009). microarray platforms were different, that microarrays were Furthermore, several putative hypoxia response elements processed at different facilities several years apart, and that (HRE) were identified in the promoters of several V-ATPases stringent criteria were used (FDR Qo0.05), a 48% retention (Supplementary Table S2). To determine whether Hif-1a rate was considered satisfactory and suggested that many contributed to V-ATPase regulation, we examined the effects other genes besides V-ATPases were bona fide targets of of both hypoxia and Hif-1a knockdown on V-ATPase expres- mTORC1. sion. As a positive control, the expression of the canonical To specifically determine the contribution of Tfeb to Hif-1 target Glut-1 was evaluated. Hypoxia stabilized Hif-1a mTORC1-induced gene expression and the LLHL pattern, protein and this was accompanied by increased expression we compared scrambled versus Tfeb shRNA-expressing of Glut-1 (Figure 3C and D). As expected, the upregulation of cells. Quite surprisingly, Tfeb knockdown markedly Glut-1 by hypoxia was markedly blunted by Hif-1a knock- decreased the number of genes with an LLHL pattern (1146 down (Figure 3C and D). We next examined V-ATPase levels. versus 424; Figure 4C). Furthermore, as visually illustrated While in Tsc2 þ / þ cells, we did not observe an increase in V- in the heatmap (Figure 4D), in 26% of LLHL probes, Tfeb ATPase expression following hypoxia (or a consistent down- knockdown resulted in a substantial ‘flattening’ of the LLHL regulation following Hif-1a knockdown), in Tsc2/ cells pattern (420% reduction in mean expression levels in there was a trend towards induction by hypoxia and this Tsc2/ MEFs by Tfeb knockdown), including several was somewhat blunted by Hif-1a knockdown, which also, in V-ATPases (Supplementary Table S3). LLHL ‘flattening’ was some cases, downregulated baseline V-ATPase expression confirmed for several V-ATPase genes by qRT–PCR levels (Figure 3D). Thus, while Hif-1a may partially contri- (Figure 4E). Interestingly, the decrease in the number of bute to V-ATPase gene regulation in Tsc2/ cells, Hif-1a does genes with an LLHL pattern after Tfeb knockdown was not seem to be the principal regulator of V-ATPase gene specific for the LLHL pattern. In fact, Tfeb knockdown expression under these conditions. resulted in an increase in the number of genes with a LLLH pattern (35 versus 323; Figure 4C; Supplementary Table S4). mTORC1-dependent V-ATPase regulation requires Tfeb These results suggest that Tfeb is an important effector A recent study showed that lysosomal biogenesis is regulated downstream of mTORC1 involved in the regulation of a by TFEB (Sardiello et al, 2009), and we noted that among the substantial number of genes. genes reported to be regulated by TFEB, there were several V-ATPases. Interestingly, the consensus sequence for TFEB Regulation of endocytosis by V-ATPases, TFEB (also called Tcfeb in the mouse), which includes the and mTORC1 E-box (Sardiello et al, 2009), overlaps with HRE (Wenger Having determined that mTORC1 regulated V-ATPase expres- et al, 2005) (Supplementary Table S2). We set out to examine sion in cells in culture and in mice and having established whether TFEB may be involved in mTORC1-dependent reg- that TFEB was required for mTORC1-induced V-ATPase ulation of V-ATPases. An antibody recognizing TFEB was expression, we sought to examine the functional significance generated in collaboration with Bethyl, and we evaluated of V-ATPase regulation by mTORC1. Because V-ATPases are the effects of Tfeb knockdown on V-ATPase expression. involved in vesicle trafficking (Marshansky and Futai, 2008),

3246 The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

100 5 +/+ A Tsc2 80 Tsc2 +/+ Rapa 80 4 Tsc2 –/– Tsc2 –/– Rapa 60 60 3

40 40 2 mRNA copies/pg RNA mRNA copies/pg RNA mRNA copies/pg RNA 20 20 1 Atp6v0c 0 0 Atp6v1b2 0 024681012 Atp6v1a 024681012 024681012 Actinomycin D treatment time (h) Actinomycin D treatment time (h) Actinomycin D treatment time (h)

B 5 5 3.5 Pol II * Pol II Pol II * IgG IgG *** 3.0 IgG 4 4 2.5 -actin -actin -actin    enrichment enrichment 3 enrichment 3 2.0

2 2 1.5 Atp6v0c Atp6v1a Atp6v1b2 1.0 1 1 normalized to normalized to normalized to 0.5 Relative Relative 0 0 Relative 0.0 –+–+Rapa –+–+Rapa –+–+Rapa Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/–

C D

+/+ –/– Tsc2 Tsc2 2 0h 3h -actin shRNA Sc Hif-1 Sc Hif-1  9h mRNA levels Hypoxia (h) 039039039039 1 Hif-1 Hif-1 normalized to 0

Atp6v1a Relative Sc Hif-1 Sc Hif-1 shRNA Tsc2 +/+ Tsc2 –/– Atp6v1b2 14 13 12 11 0h -actin 10 3h Glut-1  9 9h

mRNA levels 8 7 6 5 Tubulin Glut-1 4 3 2 normalized to 1 0

Relative Sc Hif-1 Sc Hif-1 shRNA Tsc2 +/+ Tsc2 –/–

7 3 7 0h -actin -actin -actin -actin 6 3     0h 0h 6 0h 3h

3h 3h 3h mRNA 9h mRNA 5 mRNA mRNA 9h 9h 5 9h 2 4 2 4 3 3 Atp6v0c Atp6v1a Atp6v0b Atp6v1b2 1 2 1 2 1 1 Relative Relative Relative 0 0 0 0 Relative levels normalized to levels normalized to levels normalized to levels normalized to Sc Hif-1 Sc Hif-1 shRNA Sc Hif-1 Sc Hif-1 shRNA Sc Hif-1 Sc Hif-1 shRNA Sc Hif-1 Sc Hif-1 shRNA Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Figure 3 V-ATPases are regulated transcriptionally by mTORC1 independently of Hif-1a.(A) qRT–PCR analysis of V-ATPase mRNA decay rates in the indicated cell types in low serum (n ¼ 3). (B) RNA polymerase II (Pol II) occupancy on V-ATPase gene coding sequences compared with IgG control (n ¼ 3). Western blotting (C) and qRT–PCR (D)ofTsc2 þ / þ and Tsc2/ MEFs stably transduced with a lentiviral construct with a Hif-1a (or a scrambled) shRNA sequence and exposed to hypoxia for the indicated number of hours (n ¼ 3). Error bars represent s.e.m.; *Po0.05; ***Po0.001. we evaluated the effects of mTORC1 on endocytosis. For these pathways in different cell types (Mayor and Pagano, 2007), experiments, we used albumin, the most abundant protein in albumin is taken up by MEFs through a caveolin-dependent plasma and a protein that is taken up by macrophages and mechanism (Razani et al, 2001). FITC-conjugated bovine hydrolyzed to amino acids, which are then released in the serum albumin (FITC-BSA) was efficiently taken up by circulation and are utilized by different cells (Guyton and MEFs, where it was found in punctate structures reminiscent Hall, 1996). While albumin uptake occurs through different of vesicles, which also contained V-ATPases (Figure 5A).

&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 16 | 2011 3247 Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

A 10 B 9 Sc +/+ –/– 8 A Ts c2 Ts c2 B 7 shRNA Sc A B Sc A B -actin

 6 Tfeb

mRNA levels mRNA 5 5

Tfeb Tfeb 4 Atp6v1a 3

normalized to 2 Atp6v1b2 Relative Relative 1 * * 0 *** *** Tubulin Tsc2 +/+ Tsc2 –/–

C Sc shRNA HLLL LHLL LLHL LLLH Tfeb shRNA HLLL LHLL LLHL LLLH FDR 001984 (1616) 43 (42) FDR 00644 (560) 410 (377) FDR + FC>1.5 0 0 1418 (1146) 36 (35) FDR + FC>1.5 0 0 487 (424) 351 (323)

D Scrambled Tfeb E NS +/+ −/− +/+ −/− – R – R – R – R *** *** *** ** *** 3

2 -actin -actin 2   mRNA levels mRNA levels mRNA 1 Tfeb 1 Atp6v0b normalized to normalized normalized to normalized Relative Relative

0 Relative 0 –+–+ –+–+Rapa –+–+ –+–+Rapa Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Scrambled Tfeb shRNA Scrambled Tfeb shRNA

*** *** *** ** 3 *** ***

2 -actin -actin  2  mRNA levels mRNA mRNA levels mRNA

1

Atp6v0c 1 Atp6v1a Atp6v1a normalized to normalized normalized to normalized Relative Relative 0 Relative 0 –+–+ –+–+Rapa –+–+ –+–+Rapa Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Scrambled Tfeb shRNA Scrambled Tfeb shRNA

*** *** *** 2 -actin  mRNA levels mRNA

1 Atp6v1b2 normalized to

0 Relative –+–+ –+–+Rapa Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– Scrambled Tfeb shRNA Figure 4 V-ATPase regulation by mTORC1 requires Tfeb and Tfeb is an important mediator of the changes in gene expression induced by mTORC1. (A) qRT–PCR of MEFs of the indicated genotypes stably transduced with the indicated shRNA vectors; each Tfeb shRNA (A, B) was compared with the scrambled (Sc) control by an unpaired t-test (n ¼ 3). Error bars represent s.e.m.; *Po0.05; ***Po0.001. (B) Western blot of serum-starved MEFs of the indicated genotypes. (C) Number of probes (genes) corresponding to the different patterns upregulated under one condition compared with the other three by t-tests (Qo0.05 after FDR correction) in Illumina microarrays. Data are shown also incorporating a fold change criterion (41.5-fold; FC41.5). (D) Heatmap representation of normalized expression values for all 1418 significant LLHL probes with FC41.5 in scrambled shRNA transduced MEFs compared with Tfeb shRNA transduced MEFs. Box shows probes for which mean expression levels are reduced by 420% following Tfeb knockdown in Tsc2/ MEFs. (E) qRT–PCR of Tsc2 þ / þ and Tsc2/ MEFs stably transduced with the indicated shRNA vectors and treated (or not) with rapamycin (n ¼ 3). Error bars represent s.e.m.; NS, non-significant; **Po0.01; ***Po0.001.

3248 The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

A Tsc2 +/+ Tsc2 –/– FITC-BSA Atp6v0a1 Merged FITC-BSA Atp6v0a1 Merged Vehicle Bafilomycin

B FITC-BSA incubation (min) C 03060Bafilomycin 40 ** ** Tsc2 +/+ 5 5 5 5 10 10 10 10 Tsc2 +/+ R –/– 4 4 4 4 Tsc2 +/+ 10 10 10 10 30 Tsc2 –/– R 103 103 103 103 Tsc2

0 98.6 0 98.6 0 96.9 0 94.9 20 0 103 104 105 0103 104 105 0103 104 105 0103 104 105 ** 105 105 105 105 Relative median Relative FITC-BSA uptake FITC-BSA 4 4 4 PI intensity 4 10

–/– 10 10 10 10

103 103 103 103 Tsc2

0 95.8 0 97.3 0 95.4 0 94.6 0 3 4 5 3 4 5 3 4 5 3 4 5 03060Bafi 01010 10 01010 10 01010 10 01010 10 Incubation (min) FITC intensity

D E F NS 60 ** ** 100 Untreated Scrambled Sc Sc+B V0C V1A V0+V1 Sc + Bafilomycin 50 80 ATP6V0C ATP6V1A ATP6V1A ATP6V0C + ATP6V1A 40 Tubulin 60 3 30 40 * * ** 20 2 ** median Relative FITC-BSA uptake Percent of maximum Percent of 20 10 1 0 Relative median Relative 0 uptake FITC-BSA Rapa–+ –+ –+ –+ 0 010103 4 105 Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/– FITC-BSA – + + + + + FITC intensity Bafilomycin – – + – – – Scrambled Tfeb shRNA siRNA – Sc Sc V0C V1A V0+V1 Figure 5 mTORC1 regulates endocytosis in a V-ATPase- and Tfeb-dependent manner. (A) Confocal images of serum-starved (0.1% FBS) Tsc2 þ / þ and Tsc2/ MEFs incubated with FITC-BSA and evaluated for V-ATPase colocalization following (or not) treatment with the V- þ / þ / ATPase inhibitor bafilomycin A1.(B) FACS scatterplots highlighting live (propidium iodide (PI) negative) Tsc2 and Tsc2 MEFs incubated with FITC-BSA for the indicated times; bafilomycin-treated samples shown as a control. (C) Bar graph representation of FITC-BSA incorporation in live cells pretreated (or not) with rapamycin (rapa) by reference to FITC-BSA incorporation in untreated cells (n ¼ 4). Representative FACS histogram (D) as well as bar graph representation (n ¼ 3) and western blot (E) of HeLa cells transfected with siRNA oligonucleotides targeting the V0C, V1A or both V-ATPase subunits, or a scrambled control (Sc), and incubated for 60 min with FITC-BSA; a bafilomycin A1-treated sample [B] is included as a control. (F) Bar graph representation of FITC-BSA uptake by FACS analysis in the indicated cell types in 0.1% serum and treated (or not) with rapamycin (n ¼ 3). Error bars represent s.e.m.; NS, non-significant; *Po0.05; **Po0.01.

In keeping with the idea that mTORC1 regulates endocytosis, albumin uptake (Figure 5C). Albumin uptake required FACS analyses showed higher levels of albumin uptake in V-ATPases as determined by experiments using the / þ / þ Tsc2 than Tsc2 cells (Figure 5B and C). In addition, V-ATPase inhibitor bafilomycin A1 (Bowman and Bowman, treatment of Tsc2/ cells with rapamycin downregulated 2002) (Figure 5B and C). In addition, albumin uptake was

&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 16 | 2011 3249 Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

downregulated by siRNA-mediated depletion of essential quickly Tfeb mobility was affected by rapamycin, a time

components of either the V0 or the V1 domain (alone or in course was performed. As shown in Figure 6C, rapamycin combination) in highly transfectable HeLa cervical carcinoma treatment for 1 h was sufficient to change the distribution of cells (Figure 5D and E). Tfeb. Next, we tested whether Tfeb may be similarly involved in Changes in Tfeb migration may reflect changes in phos- endocytosis. Indeed, Tfeb knockdown resulted in a statisti- phorylation, and phosphoproteomic studies have shown cally significant decrease in albumin uptake (Figure 5F). The TFEB to be phosphorylated in 410 sites (Dephoure et al, effects of Tfeb knockdown on albumin uptake in Tsc2/ cells 2008; Mayya et al, 2009). To determine whether Tfeb migra- were similar to those achieved by treatment with rapamycin tion was affected by phosphorylation, we evaluated the (Figure 5F). Taken together, these data show that mTORC1 effects of l phosphatase treatment. As shown in Figure 6D, promotes endocytosis in a Tfeb- and V-ATPase-dependent the migration of Tfeb was accelerated by treatment of manner. cell lysates with l phosphatase. Parenthetically, by compar- ison to similarly processed samples incubated without Regulation of Tfeb phosphorylation and subcellular l phosphatase for the same amount of time, phosphatase localization by the Tsc1/Tsc2 complex and rapamycin exposure not only accelerated Tfeb migration, but also led to Having established the importance of Tfeb downstream of a downregulation of total Tfeb protein levels (Figure 6D). mTORC1, we explored the mechanism whereby mTORC1 Notably, even fast-migrating Tfeb in Tsc2/ cells was subject regulated Tfeb. We observed that not only the expression to a downshift following phosphatase treatment (Figure 6D). levels of Tfeb appeared to be upregulated by mTORC1, but These results indicate that even in Tsc2/ cells, Tfeb retained also that mTORC1 affected Tfeb electrophoretic mobility on some level of phosphorylation. SDS–PAGE (Figures 4B and 6A). In Tsc2/ cells, Tfeb was We considered that the changes in Tfeb phosphorylation prominently found in a fast-migrating form(s) (Figures 4B may be linked to changes in its subcellular localization and and 6A). Treatment of Tsc2/ cells with rapamycin shifted performed subcellular fractionation experiments. Whereas Tfeb up giving rise to a pattern similar to that observed in in Tsc2 þ / þ cells, nuclear Tfeb was undetectable, Tfeb was wild-type cells (Figure 6A). Similar results were observed in found in nuclear fractions of Tsc2/ cells (Figure 6E). In the Tsc1 þ / þ and Tsc1/ cells (Figure 6B). To determine how nucleus, Tfeb appeared to be present almost exclusively in a

AB Tsc2 +/+ Tsc2 –/– Tsc1 +/+ Tsc1 –/– Rapa ––++––++Rapa

Tfeb Tfeb -S6 -S6

(S235/236) (S235/236) Tubulin Tubulin

+/+ –/– CDTsc2 –/– ETsc2 Tsc2 +/+ –/– Tsc2 Tsc2 Rapa –+–+ Rapa (h) 012346 Rapa – + –+ NCNC NCNC Tfeb λ PPase –+–+–+–+ Tfeb (short) -S6 Tfeb Tfeb (S ) (short) 235/236 Tfeb (long) S6 (long) Menin Tubulin Tubulin Tubulin

F G Tsc2 –/– –/– Tsc2 WCL Nuclei λ PPase –+ Rapa (h) 012346 012346 NCNC Tfeb (short) Tfeb Tfeb (long) Menin  -S6 (S235/236) Tubulin Menin

Tubulin

Figure 6 Coordinated regulation of Tfeb phosphorylation and nuclear localization by the Tsc1/Tsc2 complex and rapamycin. (A, B) Western blot of MEFs of the indicated genotypes in 0.1% serum treated or not with rapamycin for 24 h (as in Figure 1B). (C) Western blot of serum-starved (0.1% serum) Tsc2/ cells treated with rapamycin for the indicated times. (D) Western blot of Tsc2 þ / þ and Tsc2/ cells under serum starvation conditions treated (or not) with rapamycin and subsequently incubated, as stated, with l phosphatase. (E) Western blot of serum-starved Tsc2 þ / þ and Tsc2 / cells treated (or not) with rapamycin and subsequently fractionated into nuclear [N] or cytoplasmic [C] fractions. (F)Westernblotsof nuclear versus cytoplasmic fractions from serum-starved Tsc2 / cells incubated as indicated with l phosphatase. (G) Western blot of whole-cell lysates (WCL) or nuclei from serum-starved Tsc2/ cells treated with rapamycin for the indicated periods of time.

3250 The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al fast-migrating form (Figure 6E). To determine whether nucle- some level of phosphorylation (Figure 7E). Thus, the regula- ar Tfeb retained some level of phosphorylation, nuclear tion of TFEB phosphorylation and nuclear localization by extracts were treated with l phosphatase. As shown in TSC1/TSC2 and rapamycin was conserved across cell types. Figure 6F, this resulted in a further shift, indicating that To unequivocally establish that TFEB regulation by nuclear Tfeb was also phosphorylated. Since phosphatase TSC1/TSC2 and rapamycin was indeed mTORC1 dependent, treatment collapsed both nuclear and cytosolic Tfeb onto an we inactivated mTORC1 by knocking-down Raptor. HeLa undistinguishable band, the differences in migration likely cells were transduced with a Raptor shRNA (or a scrambled reflect differences in phosphorylation. control) and, following selection, were subjected to TSC2 Next, we sought to determine how rapamycin affected depletion using siRNA. As shown in Figure 7F, in cells nuclear Tfeb in Tsc2/ cells. Rapamycin treatment caused depleted of Raptor, TSC2 RNAi failed to accelerate TFEB a shift up in nuclear Tfeb mobility and a progressive decline mobility. In a complementary set of experiments, Raptor in nuclear Tfeb (Figure 6G). Taken together, these data siRNA (like rapamycin) retarded TFEB mobility in HeLa show that the migration and nuclear localization of Tfeb cells depleted of TSC2 (Figure 7G). Regardless of the means are coordinately regulated by Tsc1/Tsc2 and rapamycin and to deplete Raptor, Raptor inactivation prevented the accumu- suggest that mTORC1 controls the phosphorylation state and lation of faster-migrating TFEB (Figure 7F and G). Taken nuclear localization of Tfeb. together, our data indicate that mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization in multiple cell types. Regulation of TFEB phosphorylation and nuclear localization by mTORC1 To extend these studies, we examined the regulation of TFEB mTORC1-dependent nuclear localization of TFEB by mTORC1 in HeLa cells. HeLa cells were transduced with requires C-terminal serine-rich motif lentiviruses encoding a TSC2 shRNA or a scrambled control To dissect TFEB regulation by mTORC1, we first identified and, as for MEFs, cultured for 24 h in 0.1% FBS with or conditions in which ectopically expressed TFEB behaved like without rapamycin. The results were remarkably similar to endogenous TFEB. Epitope-tagged TFEB, when expressed at those observed in MEFs. TSC2 inactivation led to a downshift low levels in HeLa cells, preserved its regulation by mTORC1; in TFEB and this was reverted by rapamycin (Figure 7A it was enriched in the nuclei of TSC2-depleted cells and the and B). As in MEFs, phosphatase treatment accelerated levels were decreased by rapamycin (Figure 8A). To deter- TFEB migration even in cells depleted of TSC2 (Figure 7C). mine what regions were important for TFEB regulation by Subcellular fractionation studies showed the presence of fast- mTORC1, we examined a series of deletions (Figure 8B). migrating TFEB in nuclear fractions of cells depleted of TSC2, TFEB233–476 and TFEB290–476 behaved like full-length TFEB, but not in controls (Figure 7D) and nuclear TFEB retained indicating that the N-terminal half of TFEB was dispensable

ABHeLa CHeLa shRNA Sc TSC2 HeLa TSC2 shRNA shRNA Sc TSC2 Rapa – + – + Rapa (h) 0 1 2 3 4 6 Rapa –+–+ TSC2 λ PPase – + – +–+ –+ TFEB TFEB (short) TFEB  -S6 (S235/236)

-S6 (S ) TFEB (long) 235/236 S6

Tubulin Tubulin Tubulin

DEFGHeLa HeLa siRNA Sc TSC2 shRNA Sc TSC2 HeLa HeLa shRNA Sc Rptor Sc Rptor siRNA Sc Rptor Sc Rptor Nuclei Cytosol TSC2 shRNA TSC2 TSC2 λ shRNA Sc TSC2 Sc TSC2 PPase –+ Raptor Raptor Rapa –+ – + –+ –+ N C N C -S6K1 -S6K1 TFEB TFEB (T389) (T389) (short) (short) S6K1 S6K1 TFEB TFEB (long) TFEB TFEB (long) (short) (short) Menin Menin TFEB TFEB (long) (long) TubulinTbli Tubulin Tubulin Tubulin

Figure 7 mTORC1-dependent regulation of TFEB phosphorylation and nuclear localization. (A) Western blot of HeLa cells stably transduced with lentiviruses containing a TSC2 shRNA (or a scrambled control, Sc) and serum-starved (0.1% FBS) ± rapamycin for 24 h. (B) Western blot of serum-starved TSC2-depleted HeLa cells treated with rapamycin for the indicated hours. (C) Western blots from HeLa cells as in (A) incubated, where stated, with l phosphatase. (D) Western blots of cellular fractions from HeLa cells treated as indicated. (E) Western blot of nuclear [N] and cytoplasmic [C] fractions from TSC2-depleted serum-starved HeLa cells incubated (or not) with l phosphatase. (F) Western blot of HeLa cells stably transduced with a lentivirus encoding a Raptor shRNA (or a scrambled control, Sc) and subsequently transfected with siRNAs and serum starved. (G) Western blot of HeLa cells stably transduced with lentiviral vectors as indicated and subsequently transfected with siRNA and serum starved. Rptor, Raptor.

&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 16 | 2011 3251 Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

A B TFEB

Gln-rich Basic HLH LZ Pro-rich 476 100 233 476 80 290 476 60 321 476

40 *** 469

Percent fraction Percent 20 461

0 320 C Rapa –+–+ C/N N shRNA Sc TSC2

C 100 C/N 80 N 60 ** *** 40 * *** 20 ** Percent fraction Percent

0 Rapa –++ –+–+–+–+–+–+–+–+–+–+–+–+– shRNA Sc TSC2 Sc TSC2 Sc TSC2 Sc TSC2 Sc TSC2 Sc TSC2 Sc TSC2 TFEB (1–476) TFEB (233–476) TFEB (290–476) TFEB (321–476) TFEB (1–469) TFEB (1–461) TFEB (1–320) Growth D shRNA Sc TSC2 F Factors Rapa–+ – + TSC1 TSC2 TFEB (1–476)

GTP Rheb Rapamycin Late endosome mTOR Amino mTOR TFEB (S142A) acids Raptor GβLGRaptor βL

(Inactive) (Active)

P PP Tfeb Tfeb TFEB (5×SA)

V-ATPase

P V-ATPase TFEB (5×SD) Tfeb lysosomal genes

Nucleus

E 100 100 80 80 NS *** 60 *** *** *** *** 60 NS 40 40

20 20 NS Percent nuclear fraction 0 Percent nuclear fraction 0 Rapa–+–+ –+–+ Rapa–+–+ –+–+ shRNA Sc TSC2 Sc TSC2 shRNA Sc TSC2 Sc TSC2 TFEB (1–476) TFEB (S142A) TFEB (5×SA) TFEB (5×SD) Figure 8 mTORC1-dependent nuclear TFEB localization requires C-terminal serine-rich motif. (A) Confocal microscopy (representative images and bar graph quantification) of TFEB subcellular localization in HeLa cells stably expressing a scrambled shRNA (Sc) or a shRNA targeting TSC2 transfected with myc-tagged full-length TFEB and incubated for 24 h in 0.1% FBS with or without rapamycin. Percent of cells with cytoplasmic TFEB [C], nuclear [N] or both [C/N]. ‘*’ Illustrates statistically significant differences in nuclear TFEB (N þ C/N) between TSC2-depleted HeLa cells by comparison to the other three conditions (n ¼ 4). (B) Schematic of full-length TFEB and deletion mutants; HLH, helix-loop-helix; LZ, leucine zipper. (C) Quantification of the subcellular distribution of mutant TFEB, compared with wild-type, in HeLa cells. ‘*’ Illustrates statistically significant differences in nuclear TFEB (N þ C/N) between TSC2-depleted HeLa cells by comparison to the other three conditions for each panel (n ¼ 3). Confocal microscopy images (D) and bar graph quantification (E) of wild-type or mutant TFEB; 5 SA, S462/463/466/467/469A; 5 SD, S462/463/466/467/ 469D. Error bars represent s.e.m. (n ¼ 3). (F) Model of TFEB regulation by mTORC1. NS, non-significant; *Po0.05; **Po0.01; ***Po0.001.

3252 The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al for mTORC1-dependent nuclear localization (Figure 8C). A MEFs HeLa Within this region, mutation of a serine residue (S142) Glucose deprivation (h) 0 0.5 1 3 6 9 0 0.5 1 3 6 9 proposed to be phosphorylated by ERK2 and to regulate TFEB nuclear localization (Settembre et al, 2011) had no Tfeb appreciable effect (Figure 8D and E). Deletion of residues Grp78 1–320 resulted in a protein that was enriched in nuclei Tubulin (Figure 8C). Interestingly, deletion of just 15 amino acids Amino-acid deprivation (h) 0 0.5 1 3 6 9 0 0.5 1 3 6 9 from the C-terminus abrogated mTORC1-dependent nuclear Tfeb localization (Figure 8C). By contrast, deletion of seven amino acids had no effect (Figure 8C). These data indicated that LC3 there were critical residues between 462–469. This corre- Tubulin sponded to a serine-rich region (462SSRRSSFS469). 0.1% FBS (h) 0 0.51 3 6 9 0 0.5 1 3 6 9 To further evaluate, this serine-rich motif, we mutated all S Tfeb to A (5 SA). Interestingly, TFEB5 SA was largely cytoplas-  mic and failed to be driven into the nucleus in cells with -Erk1/2 (T183/Y185) active mTORC1 (Figure 8D and E). Thus, a serine(s) within Erk1/2 this motif is necessary for mTORC1-dependent nuclear TFEB Tubulin localization. Conversely, mutation of the same residues to aspartic acid resulted in an enrichment of TFEB in the DTT (h) 0 0.5 1 3 6 9 0 0.51 3 6 9 nucleus (Figure 8D and E). TFEB5 SD localized to the Tfeb nucleus even in cells with inactive mTORC1. Interestingly, Atf4 the percentage of cells with TFEB5 SD in the nucleus was the Tubulin same as for wild-type TFEB in mTORC1-active cells (Figure 8E; see also Figure 8A). These data show that the B MEFs HeLa × 5 4 10 106 presence of phosphomimetic residues in the serine-rich Sc Sc motif obviates the mTORC1 requirement for TFEB nuclear shTfeb A shTFEB 110 5 shTfeb B × 5 shTFEB 111 localization. The simplest explanation for these data is that 3×10 8 10 a serine(s) in the C-terminal serine-rich motif is phosphory- 6×105 5 lated in an mTORC1-dependent manner and that phosphor- 2×10 5 ylation is both necessary and sufficient to drive TFEB into the 4×10 Cell number 5 nucleus (see model in Figure 8F). 10 2×105

Complex TFEB regulation beyond mTORC1 0 0 01234 01234 by multiple signals Days Days Next, we examined TFEB in wild-type MEFs and HeLa cells Figure 9 Complex TFEB regulation by multiple signals beyond grown under standard conditions (10% FBS). TFEB was mTORC1. (A) HeLa and MEFs were starved for amino acids, glucose found in both a slow and fast-migrating form(s) in MEFs, and serum (0.1% FBS), or treated with 10 mM DTT for the indicated but predominantly in a slow migrating form(s) in HeLa cells times. (B) Graphs depicting cell counts of MEFs and HeLa cells over 4 7 (Figure 9A). We evaluated the effects of multiple interven- time (Sc, scrambled). Po10 and Po10 for Tfeb knockdown MEFs and HeLa cells, respectively, using two-factor ANOVA. Error tions on TFEB mobility. Glucose withdrawal resulted in a bars represent s.e.m. (n ¼ 3). downshift in TFEB in both MEFs and HeLa cells (Figure 9A). Amino acid withdrawal induced acute changes in TFEB migration in HeLa cells and subsequently led to the accumu- Finally, since TFEB is an oncogene (Davis et al, 2003; lation of slow migrating TFEB, which was also observed in Kuiper et al, 2003; Srigley and Delahunt, 2009), we tested MEFs (Figure 9A). The mobility of TFEB was also affected by whether TFEB was implicated in the regulation of cell pro- serum deprivation in both MEFs and HeLa cells (Figure 9A). liferation. As shown in Figure 9B, stable depletion of TFEB in Finally, changes in TFEB mobility were also induced by both MEFs and HeLa cells lowered the rates of cell expansion. treatment with a reducing agent, DTT. DTT led to a progres- sive shift in TFEB to a fast-migrating form(s) in both HeLa Discussion and MEFs (Figure 9A). Overall, these data show that TFEB is extensively and Using a robust experimental paradigm, we uncovered a novel dynamically regulated by multiple stimuli. While post-trans- regulatory network linking endocytosis to TFEB and lational modifications other than phosphorylation may be mTORC1. An unbiased gene expression analysis coupled involved in the changes observed in TFEB migration, with a design amenable to refined statistics led us to identify TFEB could be down-shifted by phosphatase treatment in a connection between mTORC1 and V-ATPase genes. Publicly every instance (data not shown). The dynamic changes in available data sets confirmed the link and experiments in TFEB mobility induced by the various interventions could not mice showed that mTORC1 regulated V-ATPase expression be explained by their expected effects on mTORC1 suggesting also in vivo. A recent study had established that TFEB that TFEB regulation involves multiple pathways and is likely regulated lysosomal biogenesis (Sardiello et al, 2009) and to be rather complex. This is consistent with the finding that among the genes regulated by TFEB we found several TFEB is phosphorylated in 410 sites (Dephoure et al, 2008; V-ATPases. These results led us to hypothesize that Mayya et al, 2009; Yu et al, 2011). V-ATPase regulation by mTORC1 may be mediated by TFEB.

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Indeed, TFEB was necessary for V-ATPase upregulation by notion, nuclear TFEB was phosphorylated. This model may mTORC1. mTORC1 regulated TFEB phosphorylation and seem at odds with the observation that nuclear TFEB is fast- nuclear localization and a C-terminal serine-rich motif was migrating and possibly hypo-phosphorylated by comparison identified that is essential for mTORC1-dependent TFEB to cytoplasmic (presumably cytosolic) TFEB. However, as our nuclear localization. Our data suggest that mTORC1 induces data suggest, nuclear TFEB may be phosphorylated on sites the phosphorylation of a serine(s) within this motif driving that are not phosphorylated on cytosolic TFEB, and thus no thereby TFEB to the nucleus. TFEB regulates the expression inferences can be drawn about the degree of phosphorylation of V-ATPases and other lysosomal genes and TFEB was based on SDS–PAGE migration. Furthermore, while we have required for mTORC1-induced endocytosis. To our knowledge identified a critical motif involved in mTORC1 regulation, this is the first study to show that endogenous TFEB is other sites in TFEB appear to be regulated by mTORC1 regulated at the subcellular localization level and that (Yu et al, 2011). mTORC1 promotes TFEB nuclear localization. These data Our results further emphasize the link between mTORC1 link an oncogenic transcription factor that is a master and the late endosome/lysosome and provide evidence for regulator of lysosomal biogenesis, TFEB, to mTORC1 and the existence of bidirectional regulatory loops. mTORC1 endocytosis. localizes to the surface of the late endosome/lysosome in a An increasing amount of evidence implicates TORC1 in highly choreographed manner (Korolchuk et al,2011; endocytosis. mTOR was identified in an RNAi screen Narita et al, 2011). The late endosome/lysosome is necessary for kinases involved in clathrin-mediated endocytosis for mTORC1 activation (Flinn et al, 2010; Li et al,2010; (Pelkmans et al, 2005) and Tsc2-deficient cells were pre- Sancak et al, 2010) and mTORC1 is reactivated under condi- viously shown to have increased fluid-phase endocytosis tions of persistent starvation following macromolecule (Xiao et al, 1997). In Drosophila melanogaster, a sensitized degradation in autolysosomes (Yu et al, 2010). However, not screen for genes that modified a tissue-specific dTOR only is mTORC1 downstream of the lysosome, but, inasmuch overexpression phenotype identified an important regulator as mTORC1 regulates TFEB and V-ATPases, mTORC1 is of endocytosis, Hsc70-4, a clathrin uncoating factor, and also upstream. mTORC1 reactivation by autolysosomes the authors showed that dTOR was involved in fat body endo- requires Spinster, a lysosomal efflux sugar transporter. cytosis (Hennig et al, 2006). Interestingly, besides V-ATPases, Spinster has been proposed to act by regulating lysosomal mTORC1 regulated the expression of multiple lysosomal pH (Rong et al, 2011), which is controlled primarily by the genes (Figure 1D; see also Supplementary Table S5). V-ATPase. Interestingly, not only has luminal pH been These effects may be mediated, at least in part, by implicated in mTORC1 regulation, but changes in cytosolic TFEB, which regulates lysosome biogenesis (Sardiello et al, pH occur in response to alterations in nutrient conditions 2009). Thus, one mechanism whereby mTORC1 and TFEB (Korolchuk et al, 2011) and have been implicated in the may affect endocytosis is by regulating V-ATPase levels regulation of lysosome topology and mTORC1 (Korolchuk and lysosomes. et al, 2011). The lysosome is the final destination not only for endoso- Recently, TFEB was found to regulate autophagy gene mal cargo but also for autophagosomes. In response expression (Settembre et al, 2011). Starvation led to ERK2 to starvation, mTORC1 is inhibited leading to the formation inactivation, which in turn, decreased TFEB phosphorylation of autophagosomes that engulf intracellular components for and increased its nuclear localization. This model is in recycling and sustenance (He and Klionsky, 2009; Kroemer principle compatible with our results. TFEB is phosphory- et al, 2010). Autophagosomes and their content are delivered lated in over 10 sites (Dephoure et al, 2008; Mayya et al, 2009; to lysosomes, which accumulate in the perinuclear area Yu et al, 2011), and mechanisms likely exist that drive (Korolchuk et al, 2011). Autophagosomes fuse with lyso- TFEB into the nucleus independently of the activation state somes to generate autolysosomes, a process that rapidly of mTORC1. However, the studies do differ in one aspect. depletes the lysosomal pool (Yu et al, 2010). The degradation Whereas mutation of the putative ERK2 phosphorylation site, of macromolecules and release of their constituents into the S142, to A is reported to be sufficient to drive TFEB into the cytosol reactivates mTORC1 (Yu et al, 2010). mTORC1 reacti- nucleus (Settembre et al, 2011), we find that, at least under vation terminates autophagy and through a poorly under- low serum conditions, the subcellular localization of TFEB is stood mechanism repletes the lysosome pool (Yu et al, 2010). not appreciably altered by a S142A mutation. However, how the pool of lysosomes is repleted is unknown. The experimental conditions studied by Settembre et al Our results suggest that lysosomal reformation in response to (2011), which involve evaluating ectopically expressed TFEB autophagy, which is mTORC1 dependent (Yu et al, 2010), may in cells in the absence of glucose, amino acids and serum, are be mediated, at least in part, by TFEB. quite different from ours. Individual deprivation of glucose, mTORC1 coordinately regulates TFEB phosphorylation and amino acids or serum has profound, dynamic and diverse nuclear localization. mTORC1 promoted TFEB nuclear loca- effects on TFEB (see Figure 9A). Given these observations lization and this process required a C-terminal serine-rich and the complexity of TFEB phosphorylation (Dephoure et al, motif. Serine substitution to non-phosphorylatable residues 2008; Mayya et al, 2009; Yu et al, 2011), how starvation of all abrogated TFEB regulation by mTORC1. More importantly, three sources will affect TFEB is unclear. Consistent with this mutation to phosphomimetic amino acids was sufficient to complexity, the changes in TFEB migration observed follow- reproduce the effect of mTORC1 on TFEB nuclear localization ing deprivation of individual factors could not be explained obviating the need for mTORC1. These data support a model by the predicted changes on mTORC1 activity. In fact, we in which mTORC1 (or an effector kinase) directly phosphor- speculate that the discovery that mTORC1 regulates TFEB ylates a serine(s) within the C-terminal serine-rich motif may have been possible only because of the tight experi- driving thereby TFEB to the nucleus. Consistent with this mental system we used.

3254 The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization Regulation of TFEB and V-ATPases by mTORC1 S Pen˜a-Llopis et al

Besides V-ATPases and lysosomal genes, many other genes mTORC1 (Duvel et al, 2010) and our results suggest that downstream of mTORC1 were regulated in a Tfeb-dependent they are affected by rapamycin to a greater extent than by manner. Approximately 25% of genes induced by mTORC1 Tsc1/Tsc2. required Tfeb. Experiments are ongoing to determine In summary, our work uncovered a novel regulatory how many of these genes are directly regulated by Tfeb. network connecting mTORC1 to a master regulator of A similar experimental setup previously identified Hif-1a as lysosome biogenesis, TFEB, which is essential for mTORC1- an mTORC1 effector (Brugarolas et al, 2003), and Hif-1a is induced endocytosis. an important mediator of a metabolic gene expression programme (Duvel et al, 2010). Along with sterol regulatory Materials and methods element-binding protein-1 (Porstmann et al, 2008), a trans- cription factor whose nuclear localization is regulated by Cell culture, drug and hypoxia treatments mTORC1, and Hif-1a, TFEB is an important mediator of MEFs Tsc2 þ / þ ; p53/ (Tsc2 þ / þ ) and Tsc2/; p53/ (Tsc2/) mTORC1 effects on gene expression. were a gift of DJ Kwiatkowski (Brigham and Women’s Hospital, Harvard Medical School, Boston, MA). Cells were grown in high The discovery that mTORC1 regulates TFEB has clinical glucose Dulbecco’s modified Eagle’s medium (Sigma) supplemen- implications. It may explain the expression of melanocyte ted with 10% FBS (HyClone) and 1% (v/v) penicillin/streptomycin lineage markers in tumours of patients with TSC (Martignoni (P/S) (Gibco) in a humidified incubator at 371C and 5% CO2.For et al, 2008), a syndrome arising from mutations in either the experiments, cells were plated to achieve B70% confluency at the time of treatment. For low-serum experiments, cells were washed TSC1 TSC2 or genes, resulting in constitutive mTORC1 activa- twice in PBS and medium was changed to the same base medium tion (El-Hashemite et al, 2003; Kenerson et al, 2007). Patients but supplemented with 0.1% FBS and cells were harvested after with TSC are predisposed to develop renal angiomyolipomas, 24 h. Where indicated, rapamycin (LC Laboratories) was used at which express melanocytic markers such as HMB45 and 25 nM in methanol and methanol was used as vehicle. Where indicated, cells were treated with actinomycin D (Sigma) at 10 mg/ Melan-A (Martignoni et al, 2008), and since expression of ml, bafilomycin A1 (LC Laboratories) at 100 nM, or DTT (10 mM). these proteins in melanomas is thought to be driven by TFEB For growth curves, cells were plated on day 0, maintained in 10% (or related family members with whom TFEB can hetero- FBS and triplicates were counted every 24 h. For hypoxia experi- 1 dimerize) (Steingrimsson et al, 2004), these markers may be ments, cells were exposed to 1% O2 and 5% CO2 at 37 Cina similarly driven by TFEB in renal angiomyolipomas. hypoxia chamber (Coy Laboratory Products). For starvation time courses, cells were washed with PBS twice and starved of amino Interestingly, TFEB functions as an oncogene in the kidney. acids (amino acid-free media), glucose (glucose-free media) or The TFEB gene is translocated in a subset of renal tumours serum (0.1% FBS) for different amounts of time. where it is subjected to a robust heterologous promoter (Davis et al, 2003; Kuiper et al, 2003). Translocation carcino- Hif-1a, Tfeb, Raptor and TSC2 shRNAs Recombinant lentiviruses were generated in HEK293T cells by mas tend also to aberrantly express melanocytic markers transient transfection using calcium phosphate with the envelope (Srigley and Delahunt, 2009), which may be similarly plasmid pMD2.G (Database ID p486), packaging plasmid psPAX2 driven by TFEB. TFEB activation may also account for the (p485) and the specific plasmid of interest. pMD2.G, psPAX2 and expression of cathepsin-K, a lysosomal protease that has been pLKO.1 or pGIPZ vectors were transfected at a ratio of 3:8:10. Supernatants were collected for 2 days, passed through a 0.45-mm recently proposed as a marker for this tumour type filter, and used to infect MEFs or HeLa cells for 24 h in medium (Martignoni et al, 2009). Interestingly, translocation carcino- containing 8 mg/ml polybrene (Sigma). MEFs and HeLa cells were mas are often recognized by immunohistochemical studies selected with 2 mg/ml puromycin for at least 1 week and HeLa cells showing high levels of nuclear TFEB (Srigley and Delahunt, were further enriched by FACS. The following vectors were used: Hif-1a 0 2009). Because TFEB nuclear localization is regulated by pLKO.1 vectors with a shRNA (5 -AGAGGTGGATATGTCTGG G-30) (p481) generously provided by LB Gardner (Nemetski and mTORC1 and as we show, sirolimus excludes TFEB from Gardner, 2007), a mouse Tfeb shRNA from Open Biosystems the nucleus, sirolimus, or its derivatives, temsirolimus and (sequence A, TRCN0000085548, 50-CGGCAGTACTATGACTATGAT- everolimus, may be effective against this tumour type. 30) (p633) or a designed Tfeb shRNA (sequence B, 50-GGCAGTAC 0 While this study focused on a small subset of genes, many TATGACTATGATG-3 ) (p652), a human TFEB shRNAs from Open Biosystems (sequence 110, TRCN0000013110, 50-GAGACGAAGGTT genes were identified whose expression was regulated CAACATCAA-30) (p700) or (sequence 111, TRCN0000013111, 50-GAA by mTORC1 not only positively but also negatively CAAGTTTGCTGCCCACAT-30) (p701), a raptor shRNA from Addgene (Supplementary Figures S8–S10; Supplementary Table S1). (Addgene plasmid 1858) (p741) or a scrambled sequence (50-GGGT 0 Moreover, genes were also found whose expression was CTGTATAGGTGGAGA-3 ) (p480). pGIPZ vectors containing a TSC2 (p496) or a scrambled (p483) shRNA were from Open Biosystems. regulated by Tsc2 independently of mTORC1 (LLHH and HHLL patterns). As previously reported (Brugarolas et al, Western blotting and antibodies 2003), this would point towards mTORC1-independent func- Cell lysates and western blotting was performed as previously tions of the Tsc1/Tsc2 complex (Supplementary Figure S8; described (Vega-Rubin-de-Celis et al, 2010), using antibodies from Supplementary Table S1). In addition, the expression of some the following sources: S6K1, phospho-S6K1 (T389), S6, phospho-S6 (S ), total 4E-BP1, phospho-4E-BP1 (T ), phospho-4E-BP1 genes appeared to be regulated by rapamycin to a signifi- 235/236 37/46 (S65) from Cell Signaling; HIF-1a, TSC1 and Menin from Bethyl cantly greater extent than by Tsc2 (HLHL and LHLH patterns) laboratories; TSC2, ATF4 and c-Myc from Santa Cruz; Raptor from (Supplementary Figure S8; Supplementary Table S1). Millipore; Erk1/2, phospho-Erk1/2 (T183/Y185) and Tubulin from Importantly, in all these settings, analyses of alternative Sigma; Cyclophilin B from Abcam; GLUT-1 from Novus Biologicals; and Grp78 from BD Biosciences. Atp6v1a and Atp6v1b2 antibodies patterns showed the associations to be statistically significant were a generous gift from D Brown (Massachusetts General (Supplementary Figure S8). Incidentally, among the genes Hospital, Boston, MA); and Atp6v0a1 and Atp6v0a3 antibodies with an HLHL pattern, there was enrichment for genes were kindly provided by V Marshansky (Massachusetts General implicated in glycolysis, pentose phosphate pathway and Hospital, Boston, MA). Tfeb antibodies were developed in collaboration with Bethyl Laboratories. fatty acid biosynthesis (Supplementary Table S5). These For the remaining Materials and methods, please see the pathways were recently reported to be regulated by Supplementary data section.

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Accession numbers EB 05556 to JCS; NIGMS 77253 to DRC; and the following Grants to Microarrays were deposited in GEO (http://www.ncbi.nlm.nih.gov/ JB: K08NS051843, RO1CA129387, RSG115739 from the ACS and a geo/) under accession numbers GSE27982 and GSE28021. BOC 5FY06582 from the March of Dimes Foundation. Microarray samples were processed at the Genomics Shared Resource of the Supplementary data Harold C Simmons Cancer Center supported in part by Supplementary data are available at The EMBO Journal Online 1P30CA142543. JB is a Virginia Murchison Linthicum Scholar in (http://www.embojournal.org). Medical Research at UT Southwestern. The content is solely the responsibility of the authors and does not represent official views from any of the granting agencies. Acknowledgements Author contributions: SP-L, SV-RC and JB designed the experi- ments. SP-L performed experiments, all statistical and microarray We thank Dr WG Kaelin Jr (Dana-Farber Cancer Institute) and the analyses and wrote an initial draft of the manuscript. SV-RC con- members of the Brugarolas laboratory for helpful discussions; MG ducted confocal microscopy and most TFEB experiments, JCS Roth, JM Abrams and MAWhite (UT Southwestern Medical Center) carried out ChIP assays under the supervision of DRC. NCW was for reading the manuscript; and C Li (Dana-Farber Cancer Institute) responsible for the mouse experiments. TATT helped with shRNA for statistical advice. We are indebted to Drs DJ Kwiatkowski cloning. LZ was involved in preliminary microarray data character- (Brigham and Women’s Hospital, Harvard Medical School, ization. X-JX provided statistical input and revised statistics. JB Boston, MA) for Tsc2-deficient MEFs and Tsc1F/F mice; LB conceived the study, was responsible for the direction of the project Gardner (New York University) for Hif1-a and scrambled shRNA and wrote the manuscript. plasmids; N Esumi (Johns Hopkins University) for TFEB plasmid; and D Brown and V Marshansky (Massachusetts General Hospital, Boston, MA) for V-ATPase antibodies. This work was supported by a fellowship of Excellence (BPOSTDOC06/004) from Generalitat Conflict of interest Valenciana (Spain) to SP-L; a fellowship from Fundacion Caja Madrid to SV-R; T32CA124334 and F32CA136087 to TATT; NIBIB The authors declare that they have no conflict of interest.

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