Current address: Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom.
In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease.
Copyright: © 2018 De Vito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files. RNAseq data are available in the ArrayExpress database under the accession number E-MTAB-6452.
Funding: This study was funded by the Ajinomoto Innovation Alliance Program, (AIAP; https://www.ajinomoto.com/en/rd/AIAP/index.html#aiap) (to M.V.S and D.G), which is a joint research initiative of Ajinomoto Co., Inc., Japan. One of the authors [M.B.] is an employee of Ajinomoto Co., and his specific roles are articulated in the ‘author contributions’ section. The commercial funder provided support in the form of salary for author [M.B.] and some of the necessary research materials (medium for cell culture), but did not have any additional role in the study design, data collection and analysis, or preparation of the manuscript, and the authors had unrestricted access to the data. Due to a confidentiality agreement, the commercial funder participated only in the decision to publish the data obtained during the study, without any restriction.
Competing interests: This study was funded by Ajinomoto Co., Inc., Japan and one of the authors [M.B.] is an employee of this commercial funder. No other employment or consultancy relationships exist with the commercial funder, and no patents, products in development, or marketed products result from this study. The authors declare that no competing interests exist and that the commercial affiliation of one of the authors does not alter the adherence of authors to all PLOS ONE policies on sharing data and materials.
In animals, excessive, insufficient, or imbalanced nutrient availability is known to strongly impact on phenotype and health, both short and long-term, and across generations [1, 2]. In particular, studies in yeast, animal models and humans have shown that reduced food intake, reducing either overall calories, or only sugars, proteins, or even single amino acids (AA), such as Methionine (Met), may extend lifespan and healthspan, and reduce the risk of cancer and other age-related diseases [3–9]. In addition, fasting or specific AA deprivation have shown potential therapeutic applications, owing to their ability to directly reduce the growth of some tumor types [10, 11], sensitize cancer cells to chemo- or immunotherapy [12, 13], and allow efficient hematopoietic stem cell engraftment . However, little is known about the specific processes and molecular mechanisms mediating the roles of nutrient restriction in human health and longevity.
A properly balanced diet in metazoans contains optimal amounts of a subset of AA, which cannot be synthetized de novo and are therefore named essential amino acids (EAAs). In humans these include Met, Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Phenylalanine (Phe), Threonine (Thr), Tryptophan (Trp), and Valine (Val), while a few others are considered as semi-essential, such as Glutamine (Gln) and Tyrosine (Tyr) [15, 16]. Consistently, EAA deprivation triggers a cell-autonomous adaptive response, characterized by extensive metabolic and gene expression modifications, implementing biosynthetic, catabolic, and plasma membrane transport processes, aimed at reconstituting the full AA complement [17, 18]. The best known and conserved pathways responding to AA deprivation are triggered by mechanistic Target of Rapamycin Complex 1 (mTORC1) and General amino acid Control Non-derepressible 2 (GCN2) protein kinases [15, 19, 20]. Activation of mTORC1 requires in particular the presence of Gln, Arg and Leu, but also Met , which activate the kinase through sensors mainly acting upstream of Rag GTPases at lysosomal membranes . In turn, mTORC1 promotes cell growth, proliferation and anabolism upon activation, and translational attenuation and autophagy upon inhibition [19, 20].
By contrast, GCN2 is activated by deprivation of any individual EAA, by means of its histidyl-tRNA synthetase-related domain, which binds uncharged tRNAs accumulating during AA limitation [23, 24]. Upon activation, GCN2 phosphorylates and inhibits its only known downstream target, namely the eukaryotic Initiation Factor 2 α (eIF2α), thereby initiating the Integrated Stress Response (ISR). This leads to attenuation of general translation, and induction of a transcriptional/translational program, aimed at increasing stress resistance and restoring cell homeostasis, by upregulating a specific subset of genes, including Activating Transcription Factor 4 (ATF4) and C/EBP-Homologous Protein (CHOP) [25–27]. Thus, inhibition of mTORC1 and activation of GCN2 by AA restriction cooperate to attenuate general translation at the initiation step, increase catabolism and turnover, and enhance stress resistance to promote adaptation . However, how these processes eventually induce protective mechanisms against the alterations associated with aging, which include pervasive epigenetic and transcriptional changes [28, 29], remains largely unknown.
We previously reported the unexpected observation that prolonged deprivation of either Tyr, or of both Methionine and Cysteine (Met/Cys), triggers the selective and reversible reactivation of exogenous transcriptional units, including plasmids, retroviral vectors and proviruses, integrated into the genome and transcriptionally repressed by defensive mechanisms against non-native DNA sequences [30, 31]. This phenomenon was observed both in HeLa epithelial and ACH-2 lymphocytic human cells, and was independent of the transgene or provirus (Ocular Albinism type 1, OA1; Green Fluorescent Protein, GFP; Lysosomal-Associated Membrane Protein 1, LAMP1; Human Immunodeficiency Virus-1, HIV-1), or of the exogenous promoter driving their transcription, either viral (cytomegalovirus, CMV; Long Terminal Repeat, LTR) or human (Phospho-Glycerate Kinase 1, PGK1; Elongation Factor-1α, EF-1α) . Furthermore, this transgene reactivation response was not reproduced by serum starvation, activation of p38, or pharmacological inhibitors of mTOR (PP242 or rapamycin), sirtuins and DNA methylation. By contrast, it was induced by pan histone deacetylase (HDAC) inhibitors, and by selective inhibitors of class II HDACs . Consistently, we found that the mechanism responsible involves epigenetic modifications at the transgene promoter, including reduced nucleosome occupancy and increased histone acetylation, and is mediated in part by reduced expression of a class II HDAC, namely HDAC4 .
These findings indicate that AA deprivation induces a specific epigenetic and transcriptional response, affecting the expression of newly-integrated exogenous transgenes and proviruses, and suggesting that endogenous sequences sharing similar structural and functional features may represent a transcriptional target as well [30, 31]. In particular, transposable elements, such as LTR-retrotransposons (or endogenous retroviruses, ERVs), are genomic “parasites” anciently-integrated into the genome, and silenced by epigenetic mechanisms of mammalian cells against the spreading of mobile elements, eventually becoming "endogenized" during evolution [32, 33]. This raises the question of whether their expression is also sensitive to AA restriction. In addition, it remains unclear whether or not the transgene reactivation response is related to specific AA deprivations, and most importantly which is the AA sensing/signaling pathway involved, in particular whether the GCN2 kinase is implicated. Thus, here we used the reactivation of silenced transgenes in cultured cells, as a model to investigate a novel molecular pathway induced by imbalanced EAA starvation, implicated in the epigenetic/transcriptional regulation of exogenous non-native DNA sequences and possibly of other endogenous anciently-integrated genomic elements.
HeLa human epithelial carcinoma, HepG2 human hepatocellular carcinoma and C2C12 mouse skeletal muscle cells were maintained in DMEM containing glutaMAX (Invitrogen) and supplemented with 10% FBS (Sigma), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), at 37°C in a 5% CO2 humidified atmosphere. Cell lines carrying integrated and partially silenced transgenes were also maintained in 600–1000 μg/ml G418.
The C2C12 cell line was provided by ATCC. HeLa and HepG2 cells were obtained by Drs. F. Blasi and G. Tonon at San Raffaele Scientific Institute, Milan, Italy, respectively, and were authenticated by Short Tandem Repeat (STR) profiling, using the Cell ID System kit (Promega), according to the manufacturer’s instructions. Briefly, STR-based multiplex PCR was carried out in a final volume of 25 μL/reaction, including 5 μL Cell ID Enzyme Mix 5X, 2.5 μL Cell ID Primer Mix 10X and 3 ng of template DNA. The thermal cycling conditions were: 1 cycle at 96°C for 2 min, followed by 32 cycles at 94°C for 30 sec, 62°C for 90 sec, and 72°C for 90 sec, and 1 cycle at 60°C for 45 sec. The following STR loci were amplified: AMEL, CSF1PO, D13S317, D16S539, D21S11, D5S818, D7S820, TH01, TPOX, vWA. Fragment length analysis of STR-PCR products was performed by Eurofins Genomics, using standard procedures of capillary electrophoresis on the Applied Biosystems 3130 XL sequencing machine, and assessment of the STR profile was performed at the online STR matching analysis service provided at http://www.dsmz.de/fp/cgi-bin/str.html.
Stable cell clones, expressing myc-tagged human OA1 (GPR143) or GFP transcripts, were generated using pcDNA3.1/OA1myc-His or pcDNA3.1/EGFP vectors . Briefly, HeLa, HepG2 and C2C12 cells were transfected using FuGENE 6 (Roche) and selected with 800, 1000, and 650 μg/ml of G418 (Sigma), respectively, which was maintained thereafter to avoid loss of plasmid integration. G418-resistant clones were isolated and analyzed for protein expression by epifluorescence and/or immunoblotting.
Full DMEM-based medium, carrying the entire AA complement, and media deprived of Met/Cys (both AAs), Met (only), Cys (only), Alanine (Ala), Thr, Gln, Val, Leu, Tyr, Trp, Lys and His were prepared using the Nutrition free DMEM (cat.#09077–05, from Nacalai Tesque, Inc., Kyoto, Japan), by adding Glucose, NaHCO3, and either all 20 AAs (for full medium) or 18–19 AAs only (for deprivations of two-one AAs). Single AAs, Glucose, and NaHCO3 were from Sigma. Further details and amounts utilized are indicated in S1 Table. All media were supplemented with 10% dialyzed FBS (Invitrogen), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and G418 as required. HBSS was from Invitrogen. Cells were seeded at 10–30% of confluency; cells to be starved for 48 h were plated 2–3 times more confluent compared to the control. The following day, cells were washed and cultured in the appropriate medium, with or without EAA, for 24–48 h.
L-Histidinol (HisOH), PP242, Integrated Stress Response Inhibitor (ISRIB), SP600125, Cycloheximide (CHX) were from Sigma; Salubrinal was from Tocris Bioscience; U0126 was from Promega. Drugs were used at the following final concentrations: HisOH at 4–16 mM; PP242 at 1–3 μM; ISRIB at 100 nM; SP600125 at 20 μM in HepG2 cells and 50 μM in HeLa cells; Cycloheximide (CHX) at 50 ug/ml in HepG2 cells and 100 ug/ml in HeLa cells; Salubrinal at 75 μM; U0126 at 50 μM. Vehicle was used as mock control. Treatments with drugs to be tested for their ability to inhibit transgene reactivation (ISRIB, SP600125 and U0126) were initiated 1h before the subsequent addition of L-Histidinol (ISRIB) or the subsequent depletion of Met/Cys (SP600125 and U0126).
Total RNA was purified using the RNeasy Mini kit (Qiagen), according to manufacturer’s instructions. RNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific). Equal amount (1 μg) of RNA from HeLa, HepG2 and C2C12 cells was reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) using oligo-dT as primers, and diluted to 5 ng/μl. The cDNA (2 μl) was amplified by real-time PCR using SYBR green Master Mix on a Light Cycler 480 (Roche), according to manufacturer’s instructions. The thermal cycling conditions were: 1 cycle at 95°C for 5 min, followed by 40–45 cycles at 95° for 20 sec, 56° for 20 sec and 72° for 20 sec. The sequences, efficiencies and annealing temperatures of the primers are provided in S2 Table. Data were analyzed with Microsoft Excel using the formula EtargetΔct target (control-sample) /EreferenceΔct reference (control-sample) . Reference genes for normalizations were ARPC2 (actin-related protein 2/3 complex, subunit 2) for HeLa and HepG2 cells; and Actb (actin beta) for C2C12 cells, unless otherwise indicated.
siRNA (Mission esiRNA, 200 ng/μL; Sigma) against ATF4 and GCN2 were designed against the targeted sequences NM_001675 and NM_001013703, respectively. Cells seeded in 6-well plates were transfected with 1 μg of siRNAs and 5 μL of Lipofectamine 2000 (Invitrogen), following manufacturer’s instructions, at day 1 post-plating for ATF4 and at day 1 and 2 post-plating for GCN2. At day 2 (ATF4) or 3 (GCN2) post-plating, cells were washed and cultured in medium in the absence or presence of HisOH 4 mM for 6 h. siRNAs against RLuc (Sigma), targeting Renilla Luciferase, were used as negative control. For CRISPR/Cas9 experiments, we used the “all-in-one Cas9-reporter” vector, expressing GFP (Sigma), which is characterized by a single vector format including the Cas9 protein expression cassette and gRNA (guide RNA). GFP is co-expressed from the same mRNA as the Cas9 protein, enabling tracking of transfection efficiency and enrichment of transfected cells by fluorescence activated cell sorting (FACS). The human U6 promoter drives gRNA expression, and the CMV promoter drives Cas9 and GFP expression. The oligonucleotide sequences for the three gRNAs targeting GCN2 exon 1 or 6 are listed in S2 Table. We transfected HeLa and HepG2 cells with these plasmids individually (one plasmid one guide) and sorted the GFP-positive, transfected cells by FACS. Screening GCN2-KO clones was performed by western blotting. In the case of HepG2-OA1 cells, two rounds of selection were necessary to obtain three GCN2-KO clones by using a guide RNA against exon 1. Compared to the original HepG2-OA1 cell line and to the clone resulting from the first round of selection (185#27), the selected clones E23, F22 and F27 showed a very low amount—if any—of residual GCN2 protein (see results).
Genomic DNA of HeLa and HepG2 cells was purified using DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer’s instructions. DNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific). PCR conditions for amplification of GCN2 exon 1 and 6 were as follows: 1 cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 40 sec, 56°C for 40 sec, and 72°C for 40 sec; and a final extension step of 5 min at 72°C. The primer sequences are provided in S2 Table.
For OA1, western immunoblotting was carried out as described . For GCN2, cells were lysed in RIPA buffer, boiled at 95°C for 5 min and resolved on a 7.5% polyacrylamide gel; immunoblotting was then performed following standard procedures. Primary Abs were as follows: anti-human OA1, previously developed by our group in rabbits ; anti-GCN2 (Cell Signaling, Cat. #3302).
Statistical analyses were performed using Microsoft Excel for Mac (version 15.32, Microsoft) for Student’s t-test; or GraphPad Prism (version 5.0d for Mac, GraphPad Software, Inc.) for one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s multiple comparisons post-tests. T-test was used when only two means, typically sample versus control, were compared, as specified in the figure legends. One way ANOVA was used for multiple comparisons, followed by either a Dunnett’s (to compare every mean to a control mean), or a Tukey’s (to compare every mean with every other mean) post-test, by setting the significance level at 0.05 (95% confidence intervals). Both tests compare the difference between means to the amount of scatter, quantified using information from all the groups. Specifically, Prism computes the Tukey-Kramer test, allowing unequal sample sizes. P values in Figures are generally referred to comparison between a sample and the control (full medium/mock), and are indicated as follows: *P<0.05, **P<0.01, ***P<0.001. Comparisons not involving the control are similarly indicated, by a horizontal line at the top of the graphs, encompassing the two samples under analysis. Additional details regarding the specific experiments are reported in the Figure Legends.
To examine the expression behavior of genomic repeats upon AA starvation, we performed a transcriptomic analysis taking advantage of an intramural sequencing facility. HeLa-OA1 cells were cultured in normal medium (for 6-30-120 hours) or in absence of Met/Cys (for 6-15-30-72-120 hours). Total RNA was prepared using Trizol (Sigma) to preserve transcripts of both small and long sizes (from Alu, of about 0.3 kb, to Long Interspersed Nuclear Elements, LINEs, and ERVs, up to 6–8 kb long), DNase treated to avoid contamination of genomic DNA, and processed for NGS sequencing by Ovation RNA-Seq System V2 protocol and HiSeq 2000 apparatus. Raw sequence data (10–20 M reads/sample) were aligned to the human genome (build hg19) with SOAPSplice . Read counts over repeated regions, defined by RepeatMasker track from UCSC genome browser , were obtained using bedtools suite . Normalization factors and read dispersion (d) were estimated with edgeR , variation of abundance during time was analyzed using maSigPro package , fitting with a negative binomial distribution (Θ = 1/d, Q = 0.01), with a cutoff on stepwise regression fit r2 = 0.7. Read counts were transformed to RPKM for visualization purposes. The expression of the OA1 transgene and HDAC4, which are progressively up- and down-regulated during starvation, respectively , were used as internal controls.
For genomic repeat analysis, reads belonging to repetitive elements were classified according to RepeatMasker and assigned to repeat classes (total number in the genome = 21), families (total number in the genome = 56) and finally subfamilies (total number in the genome = 1396), each including a variable number of genomic loci (from a few hundred for endogenous retroviruses, up to several thousand for Alu). Repeat subfamilies were then clustered according to their expression pattern in starved vs control cells, by maSigPro using default parameters, and repeats classes or families that are significantly enriched in each cluster, compared to all genomic repeats, were identified by applying a Fisher Exact test (using scipy.stats, a statistical module of Python). Alternatively, differentially expressed repeat subfamilies were identified by averaging three time points of starvation (15-30-72 h) and controls. Repeats significantly up- or downregulated (104 and 77, respectively) were selected based on a P value <0.05 (unpaired two-tailed Student’s t-test, assuming equal variance), and analyzed for their class enrichment by a Fisher Exact test as described above.
For gene set enrichment analysis of Met/Cys deprived vs control HeLa cells, differentially expressed genes were selected considering three time points of starvation (15-30-72 h) and controls, based on a P value <0.05 (unpaired two-tailed Student’s t-test, assuming equal variance) and a fold change >2. This led to a total of 2033 differentially expressed genes, 996 upregulated and 1037 downregulated. The enrichment analysis was performed separately for up and down regulated genes, or with all differentially expressed genes together (both), using the KEGG database. The analysis was performed with correction for the background of all expressed genes (about 13600 genes showing an average expression over 3 starvation and 3 control samples of at least 5 counts) and by using default parameters (adjusted P value and q-value cut-off of <0.05 and 0.2, respectively). Differentially expressed genes were also selected considering all starvation time points, as with genomic repeats, by maSigPro using default parameters, and a fold change of at least 1.5, leading to similar enrichment results (not shown). RNAseq gene expression data are available in the ArrayExpress database under the accession number E-MTAB-6452.
To provide proof-of-principle that AA starvation may affect the expression of transposable elements, we performed an RNAseq analysis of the previously described HeLa-OA1 cells, carrying an integrated and partially silenced OA1 transgene . Since the reactivation of the transgene by starvation is a progressive phenomenon , we performed a time-course experiment, where each time point represents one biological sample, rather than a biological triplicate of a single time point. To this aim, cells were cultured either in normal medium, or in absence of Met/Cys for different time points (6-15-30-72-120 hours), resulting in the progressive upregulation of the OA1 transgene during starvation (Fig 1A and 1B), consistent with previously published results . The expression of genomic repeats was determined according to RepeatMasker annotation and classification into classes, families, and subfamilies. Repeat species were then subjected to differential expression and enrichment analyses in starved vs control conditions. Out of 1396 annotated repeat subfamilies, 172 species displayed a differential expression profile during starvation.
Fig 1. Exogenous transgene and endogenous retroviruses are upregulated in Met/Cys-deprived HeLa cells.
(A,B) Exogenous integrated transgene (OA1) mRNA abundance in HeLa-OA1 cells, cultured in Met/Cys-deprived medium for the indicated time points, and analyzed by RNAseq (A), or RT-qPCR (B), compared to full medium. Data represent RPKM (A), or mean ± SD of 2 technical replicates, expressed as fold change vs. control (full medium at 6 h = 1) (B). (C) Clustering of 172 genomic repeat subfamilies, differentially expressed upon starvation, according to their expression profile. (D) Class distribution of repeat subfamilies belonging to differential expression clusters, compared to all genomic repeat subfamilies (first column). Class DNA includes DNA transposons; SINE includes Alu; LINE includes L1 an L2; LTR includes endogenous retroviruses and solitary LTRs; Satellite includes centromeric acrosomal and telomeric satellites; Others includes SVA, simple repeats, snRNA, and tRNAs. LTR-retroelements are significantly enriched among repeats that are upregulated upon starvation, while LINEs are significantly enriched among repeats that are downregulated. *P<0.05, ***P<0.001 (Fisher exact test).
As shown in Fig 1C, the clustering of differentially expressed repeats, according to their expression pattern, reveals profiles comparable to the behavior of the transgene in the same conditions, i.e. upregulation upon starvation and no change in regular medium (Cluster 1 and 2). In particular, Cluster 1 contains sequences that, similarly to the OA1 transgene, are progressively upregulated upon starvation (Fig 1A and 1C) , while Cluster 2 contains sequences that are upregulated at early time points. Interestingly, repeat families that are significantly enriched in these two clusters belong mostly to the group of LTR-retrotransposons, including ERV1, ERVK, ERVL, ERVL-MaLR and other LTR sequences (Fig 1D; S1A and S2A Figs). By contrast, DNA transposons (such as TcMar-Tigger) and L1 non-LTR retrotransposons are enriched among repeats that are downregulated during starvation, particularly at late time points (Clusters 3 and 4) (Fig 1D; S1A and S2A Figs). Consistent results were obtained by selecting significantly up- or downregulated genomic repeats (overall 181 species), based on their average expression out of three time points of starvation (15-30-72 h, when the transgene upregulation is more homogeneous) and controls, and on a P value <0.05 (S1B and S2B Figs). These findings suggest that EAA starvation induces genome-wide effects involving repetitive elements, and that—among major repeat classes—it upregulates in particular the expression of ERVs.
In addition, to obtain a general overview of main gene pathways changing their expression together with the transgene during AA starvation, we performed gene expression and enrichment analyses of regular genes, by considering three time points of starvation (15-30-72 h) and controls. Differentially expressed genes were selected based on a P value <0.05 and a fold change between means of at least 2, and analyzed with the EnrichR tool . As shown in Fig 2 and S1 File, enrichment analyses against the KEGG and Reactome databases reveals a predominance of downregulated pathways, namely ribosome and translation, proteasome, AA metabolism, oxidative phosphorylation and other pathways related to mitochondrial functions, which are affected in Huntington, Alzheimer and Parkinson diseases (http://www.genome.jp/kegg/pathway.html). In particular, a large fraction of ribosomal protein mRNAs is downregulated upon Met/Cys starvation (Fig 2A and 2C; S1 File), consistent with the notion that their genes–despite being scattered throughout the genome—are coordinately expressed in a variety of conditions . This reduced expression may depend on multiple pathways that control ribosome biogenesis in response to external stimuli, including the downregulation of Myc activity , the downregulation of mTORC1 [42, 44], or possibly the activation of the ISR, as described in yeast . By contrast, upregulated genes show a significant enrichment for transcription and gene expression (Fig 2B). Similar results were obtained by the Gene Ontology Biological Process (GO-BP) database (S1 File), overall indicating a general downregulation of translation and metabolism, and upregulation of transcription, during the time interval of Met/Cys starvation corresponding to the transgene upregulation.
Fig 2. Gene set enrichment analysis of Met/Cys-deprived HeLa cells.
Differentially expressed genes between three time points of starvation (15-30-72 h) and controls were selected based on a P value <0.05 and a fold change of at least 2, leading to a total of 996 upregulated, and 1037 downregulated genes. The enrichment analysis was performed separately for up and down regulated genes, using the EnrichR tool and the KEGG (A) and REACTOME (B, C) databases. Ranking is based on the combined score provided by EnrichR, and categories are displayed up to 20 items with an Adjusted P value <0.05. No significant categories were found with upregulated genes against the KEGG database. All data are shown in S1 File. The enrichment analysis using all differentially expressed genes together did not reveal any additional enriched process.
To characterize the pathway leading to the reactivation of silenced transgenes, we used HeLa-OA1 and HeLa-GFP cells, as described . In addition, to test cell types relevant for AA metabolism, such as liver and muscle, we generated clones of HepG2 human hepatoma and C2C12 mouse skeletal muscle cells, stably transfected with plasmids for OA1 and GFP transgenes, respectively (HepG2-OA1 and C2C12-GFP cells; endogenous OA1 is not expressed in any of these cell types). In all cases, the integrated transgenes are under the control of the CMV promoter in the context of a pcDNA3.1 plasmid, are partially silenced, and can be efficiently upregulated by HDAC inhibitors (trichostatin A, TSA; ref.  and S3A, S3B and S4A Figs), indicating that their expression is controlled at least in part by epigenetic mechanisms, as previously described .
To establish whether the reactivation response results from the shortage of specific AAs only, such as Met/Cys, or it is triggered by any AA deprivations, we cultured HeLa-OA1, HeLa-GFP, HepG2-OA1 and C2C12-GFP cells for 24–48 hours with a battery of media deprived of EAAs or semi-EAAs, including Met/Cys, Thr, Gln, Val, Leu, Tyr, Trp, Lys, and His. As negative controls, cells were cultured in full medium, carrying the entire AA complement, and in a medium deprived of Ala, a non-essential AA. The expression of the transgene transcript was then evaluated by RT-qPCR. As shown in Fig 3, and in S3C and S4B Figs, most EAA-deficiencies induced reactivation of the OA1 or GFP transgenes in all four cell lines, with the notable exception of Trp deprivation, which consistently resulted in no or minimal reactivation of the transgenes. Indeed, despite some variability, Met/Cys deficiency, but also Thr, Val, Tyr, and His deprivation always gave an efficient response, while Leu, Gln and Lys elicited evident responses in some cases, but not in others. Depletion of Phe gave results comparable to Tyr deprivation, however it significantly altered multiple reference genes used for normalization and therefore was eventually omitted from the analysis (not shown). Finally, in the above experiments we used a combined Met/Cys deficiency, to avoid the potential sparing of Met by Cys  and for consistency with our previous studies . Nevertheless, the analysis of single Met or Cys starvation, both at the protein and transcript levels, revealed an exclusive role of Met deprivation in transgene reactivation, consistent with the notion that Cys is not an EAA (S3D and S3E Fig).
Fig 3. EAA deprivation induces reactivation of silent transgenes in HeLa and HepG2 cells.
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in various AA-deprived media for 48 h and 24 h, respectively, compared to full medium. Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. control (full medium = 1). *P<0.05, **P<0.01, ***P<0.001 (one way ANOVA, followed by Dunnett’s post-test vs. full medium).
Collectively, these results indicate that transgene reactivation by EAA starvation is reproducible with most EAAs, shared by different cell types (epithelium, liver, and skeletal muscle), and conserved in different mammalian species (human, mouse).
mTORC1 inhibition and GCN2 activation trigger the best-known signaling pathways responding to AA starvation . We previously showed that inhibition of mTORC1 is not sufficient to reproduce transgene reactivation in HeLa cells . By contrast, the involvement of GCN2 and the ISR, including the downstream effectors ATF4 and CHOP, has never been tested. In addition, this pathway has been typically assessed in transient assays, lasting for a few hours, which may not be comparable with the prolonged starvation conditions necessary to reactivate the transgene expression (at least 15–24 h). Thus, we tested whether CHOP expression was upregulated upon incubation of HeLa-OA1, HepG2-OA1 and C2C12-GFP cells in media deprived of different EAAs for 24–48 h.
As shown in Fig 3 and S4B Fig, we found that CHOP expression is increased in all EAA-starvation conditions, but not in the absence of Ala, in all tested cell lines. Similar, yet less pronounced, results were obtained with ATF4, consistent with the notion that activation of this transcription factor is mainly mediated by translational upregulation (not shown) [15, 26]. However, the upregulation of CHOP does not parallel quantitatively that of the transgene, neither appears sufficient to induce it. In fact, CHOP is highly upregulated even upon Trp starvation, which consistently results in no or minimal reactivation of the transgenes (compare CHOP with OA1 or GFP expression; Fig 3 and S4B Fig). Thus, while the ISR appears widely activated upon EAA starvation, the upregulation of its downstream effector CHOP only partly correlates with transgene reactivation and may not be sufficient to induce it.
The activation of the ISR upon AA starvation suggests that GCN2 may be involved in the transgene reactivation response. Therefore, we tested whether direct pharmacological activation of this kinase is sufficient to trigger the transgene reactivation similarly to starvation. In addition, we used pharmacological inhibitors of mTOR to corroborate previous negative results in HeLa cells  in the other cell lines under study. To this aim, HeLa-OA1 or GFP, HepG2-OA1 and C2C12-GFP cells were cultured in the presence of different concentrations of PP242 (mTOR inhibitor) or L-Histidinol (GCN2 activator, inhibiting tRNAHis charging by histidyl-tRNA synthetase), either alone or in combination for 24 h, compared to Met/Cys-deprived and full medium. As shown in Fig 4 and S5 Fig, while inhibition of mTORC1 consistently leads to minor or no effects, in agreement with previous findings , treatment with L-Histidinol results in efficient reactivation of the transgene in HepG2-OA1 and C2C12-GFP cells, but not in HeLa cells.
Fig 4. mTOR inhibition and GCN2 activation differently affect transgene expression in HeLa and HepG2 cells.
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in Met/Cys-deprived medium, or in the presence of PP242 (mTOR inhibitor; 1–3 μM) or L-Histidinol (HisOH, GCN2 activator; 4–16 mM), either alone or in combination for 24–48 h, compared to full medium. Mean ± SEM of 4 (A) or 3 (B) independent experiments. Data are expressed as fold change vs. control (full medium = 1). *P<0.05, **P<0.01, ***P<0.001 (one way ANOVA, followed by Dunnett’s post-test vs. full medium). PP-1 and PP-3, PP242 at 1 and 3 μM, respectively; HisOH-4 and HisOH-16, L-Histidinol at 4 and 16 mM, respectively.
Specifically, L-Histidinol is not effective in HeLa-OA1 and HeLa-GFP cells, either alone or in combination with PP242 (Fig 4A and S5A Fig), or by using different concentrations of the drug, with or without serum (not shown). In these cells, L-Histidinol appears also unable to trigger the ISR, as indicated by lack of CHOP upregulation, possibly due to their different sensitivity to the drug. These findings are consistent with previous reports, describing the use of L-Histidinol in HeLa cells in conditions of low His concentration in the culture medium , which would resemble AA starvation in our system and therefore may not be applicable. Thus, even though the amount of the amino alcohol was adapted to exceed 20 to 80 times that of the amino acid, as described , HeLa cells may be resistant or able to compensate.
In contrast, in other cell types, L-Histidinol has been utilized in regular DMEM, to mimic the AA response triggered by DMEM lacking His [48, 49]. Consistently, in HepG2-OA1 cells, L-Histidinol is sufficient to elicit extremely high levels of transgene reactivation, and its combination with PP242 results in additive or even synergistic effects, possibly due to an indirect effect of mTOR inhibition on GCN2 activity (Fig 4B) [50, 51]. Similarly, C2C12-GFP cells efficiently reactivate the transgene upon treatment with L-Histidinol, but not PP242 (S5B Fig). However, differently from HepG2-OA1 cells, simultaneous treatment of C2C12-GFP cells with L-Histidinol and PP242 does not lead to synergistic effects. Consistent with stimulation of the ISR, CHOP and to a minor extent ATF4 are upregulated by L-Histidinol in both cell lines, yet their expression levels show only an incomplete correlation with those of the transgene (Fig 4B, S5B Fig, and not shown).
The finding that GCN2 activation by L-Histidinol is sufficient to reactivate the transgenes in both HepG2-OA1 and C2C12-GFP cells pointed to this kinase, and to the downstream ISR, as the pathway possibly involved in the EAA starvation response. Thus, we investigated whether the ISR is sufficient to trigger upregulation of the OA1 transgene in HepG2-OA1 cells by pharmacological means. As CHOP expression does not correspond quantitatively and is not sufficient to induce transgene reactivation, we tested the role of the core upstream event of the ISR, namely the phosphorylation of eIF2α , which can be induced by pharmacological treatments, independent of GCN2 (Fig 5A). To this aim, we used Salubrinal, a specific phosphatase inhibitor that blocks both constitutive and ER stress-induced phosphatase complexes against eIF2α, thereby increasing its phosphorylation . We found that, while the ISR is activated upon Salubrinal treatment, as shown by increased CHOP expression, it does not induce OA1 transgene reactivation (Fig 5B).
Fig 5. The ISR is neither sufficient nor necessary to induce transgene reactivation in HepG2 cells.
(A) Schematic representation of GCN2 activation by AA starvation, resulting in phosphorylation of eIF2a and initiation of the downstream ISR. In addition to GCN2, the ISR may be activated by other eIF2a kinases (PKR, HRI and PERK; not shown in the picture). (B) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 24 h with Salubrinal (a drug that induces the ISR by inhibiting the dephosphorylation of eIF2α; 75 μM), compared to full medium. Mean ± range of two experiments. Data are expressed as fold change vs. control (DMEM = 1). *P<0.05 (paired two-tailed Student’s t-test vs. control). (C) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM), in the absence or presence of ISRIB (a drug that bypasses the phosphorylation of eIF2α, inhibiting triggering of the ISR; 100 nM). Mean ± range of two experiments. Data are expressed as fold change vs. control (DMEM = 1). **P<0.01, ***P<0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). (D) Relative transgene (OA1) and ATF4 mRNA abundance in HepG2-OA1 cells transfected with control (CTRL) or anti-ATF4 siRNAs, and incubated in the presence or absence of L-Histidinol (HisOH, GCN2 activator; 4 mM) for 6 h. Mean ± range of two experiments. Data are expressed as fold change vs. control (w/o HisOH = 1, top; control siRNA = 1, bottom). *P<0.05 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated).
To test whether the ISR is necessary to trigger the transgene response to L-Histidinol, we used the chemical compound ISRIB, which inhibits the activation of the ISR, even in the presence of phosphorylated eIF2α, likely by boosting the activity of the guanine-nucleotide exchange factor (GEF) for eIF2α, namely eIF2B [53, 54]. HepG2-OA1 cells were stimulated with L-Histidinol, either in the presence or absence of ISRIB. As shown in Fig 5C, while the expression of CHOP is inhibited by ISRIB, as expected, the reactivation of the OA1 transgene is not affected. In addition, knockdown of the closest eIF2α downstream effector ATF4 by siRNAs does not interfere with the reactivation of the OA1 transgene by L-Histidinol (Fig 5D). Together, these data suggest that eIF2α phosphorylation and the downstream ISR pathway are neither sufficient nor necessary to induce transgene reactivation.
To definitively establish if GCN2 is necessary to trigger the transgene reactivation response to EAA starvation, we directly suppressed its expression by CRISPR/Cas9-mediated knock-out (KO). We generated three independent GCN2-KO clones from the parental HeLa-OA1 cell line, by using three different guide RNAs, two against exon 1 (clones 183#11 and 185#5), and one against exon 6 (clone 239#1) of the GCN2 gene. Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone 183#11, and on both alleles of exon 6 in clone 239#1; by contrast, clone 185#5 showed multiple alleles in exon 1, consistent with the presence of two cell populations, and was not characterized further at the genomic level (S6 Fig). None of these clones express GCN2 at the protein level, as shown by immunoblotting (Fig 6A). To test the GCN2-KO cells for their ability to respond to EAA starvation, parental HeLa-OA1 cells and the three GCN2-KO clones were cultured in media deprived of Met/Cys or Thr (corresponding to the most effective treatments in this cell line; see Fig 3A) for 24–48 h and transgene expression was assessed by RT-qPCR. We found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, thus excluding that this kinase is necessary for the response to EAA starvation in HeLa-OA1 cells (Fig 6B and 6C).
Fig 6. GCN2 knockout does not interfere with transgene reactivation in HeLa cells.
(A) Immunoblotting of protein extracts from the HeLa-OA1 parental cell line and GCN2-KO clones 183#11, 185#5 and 239#1, immunodecorated with anti-GCN2 antibody. Arrow, GCN2 specific band. Ponceau staining was used as loading control. (B, C) Relative transgene (OA1) mRNA abundance in HeLa-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or Thr (C) deprived medium for 24 h or 48 h, respectively, compared to full medium. Mean ± SD of 3 technical replicates from 1 experiment. Data are expressed as fold change vs. control (full medium = 1). Since independent clones may display variable reactivation responses (e.g. due to different levels of transgene expression in basal conditions), the results are not shown as means of the three clones, but as separate replicates.
Similarly, we generated GCN2-KO clones from the parental HepG2-OA1 cell line by the same strategy. By using a guide RNA against exon 1 of the GCN2 gene, we obtained three independent GCN2-KO clones, namely E23, F22 and F27. Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone F27 (S7 Fig) and all three clones showed a very low amount—if any—of residual GCN2 protein, compared to the original HepG2-OA1 cell line (Fig 7A). To assess the ability of GCN2-KO cells to reactivate the transgene upon starvation, we cultured parental HepG2-OA1 cells and the three GCN2-KO clones in media deprived of Met/Cys or His (corresponding to the most effective treatments in this cell line; see Fig 3B) for 24 h, and evaluated the transgene expression by RT-qPCR. As shown in Fig 7B and 7C, we found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, as in HeLa cells. To further confirm this result, we knocked-down GCN2 by RNA interference (RNAi), and incubated the cells with or without L-Histidinol for 6 h. As shown in Fig 8, treatment of HepG2-OA1 cells with L-Histidinol results in efficient transgene reactivation, even upon significant GCN2 downregulation, both at the mRNA and protein levels. Taken together, these data strongly support the conclusion that GCN2 is not necessary for transgene reactivation in response to EAA starvation, either in HeLa or in HepG2 cells.
Fig 7. GCN2 knockout does not interfere with transgene reactivation in HepG2 cells.
(A) Immunoblotting of protein extracts from the HepG2-OA1 parental cell line and GCN2-KO clones 185#27, E23, F22, F27, immunodecorated with anti-GCN2 antibody. Clone 185#27 results from the first round of selection, and was used to generate clones E23, F22, F27. Arrow, GCN2 specific band. For GCN2 protein quantification, Ponceau staining was used as loading control and data are expressed as fold change vs. parental cell line (= 1). (B, C) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or His (C) deprived medium for 24 h, compared to full medium. Mean ± SD of 3 technical replicates from 1 experiment.

Question: Is the ISR necessary for transgene reactivation?
Answer: No, it is not necessary.