mTOR inhibition and GCN2 activation differently affect transgene expression in HeLa and HepG2 cells.

<p>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.</p

GCN2 knockout does not interfere with transgene reactivation in HepG2 cells.

<p>(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. 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. The greater reactivation of the transgene in the HepG2 clones (particularly upon Met/Cys deprivation) depends on the lower expression level of the transgene in basal conditions, probably secondary to subculturing, resulting in a higher starved vs. control ratio.</p

The ISR is neither sufficient nor necessary to induce transgene reactivation in HepG2 cells.

<p>(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).</p

GCN2 knockdown does not interfere with transgene reactivation in HepG2 cells.

<p>(A, B) Downregulation of GCN2 protein by RNAi, shown by immunoblotting (A) and quantification (B) of protein extracts from HepG2-OA1 cells, transfected with control or anti-GCN2 siRNAs, and incubated with anti-GCN2 antibody. Arrow, GCN2 specific band; asterisk, non-specific signal detected by the Ab. Ponceau staining was used as loading control. Data are expressed as fold change vs. control siRNA (siRNA CTRL = 1). (C) Relative GCN2 mRNA abundance in HepG2-OA1 cells transfected with control or anti-GCN2 siRNAs. Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. control siRNA (siRNA CTRL = 1). ***P<0.001 (paired two-tailed Student’s t-test vs. control). (D) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells transfected with control or anti-GCN2 siRNAs and incubated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM). Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. untreated control (w/o HisOH = 1). *<i>P</i><0.05, ***<i>P</i><0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). (E) Schematic representation of the experiment.</p

Exogenous transgene and endogenous retroviruses are upregulated in Met/Cys-deprived HeLa cells.

<p>(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. *<i>P</i><0.05, ***<i>P</i><0.001 (Fisher exact test).</p

Transgene reactivation is abolished by MAPK inhibitors, and is induced by ribosomal inhibitors.

<p>(A) Relative transgene (OA1) mRNA abundance in HeLa-OA1 and HepG2-OA1 cells cultured in full medium or Met/Cys-deprived medium, in the presence or absence of inhibitors for MEK1/2 (U0126; 50 μM), and JNK1/2/3 (SP600125; 20–50 μM) for 24 h. For HeLa-OA1 cells data represent the mean ± SEM of 4 (CTRL and SP600125), or the mean ± range of 2 (U0126) independent experiments; for HepG2-OA1 cells, data represent the mean ± SEM of 3 independent experiments. Results are expressed as fold change vs. control (full medium = 1). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). Reference genes for qPCR: ACTB (actin beta; HeLa) and ARPC2 (HepG2). (B) Relative transgene (OA1) mRNA abundance in HeLa-OA1 and HepG2-OA1 cells cultured in the presence of CHX (protein elongation inhibitor; 50–100 ug/ml) for different time points, as indicated, compared to untreated control. For HeLa-OA1 cells, data represent the mean ± SEM of 3 independent experiments; for HepG2-OA1 cells, data represent the mean ± SEM of 4 (6 h), or the mean ± range of 2 (16 h) independent experiments. Results are expressed as fold change vs. control (mock = 1). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). Since the expression of reference genes used for normalizations change considerably upon CHX treatment (particularly at 15–16 h), the data presented are not normalized. However, comparable results were obtained by ARPC2 normalization. (C) Model for the transgene reactivation response to EAA starvation. EAA deficiency inhibits mTORC1 and activates GCN2, which attenuate general translation at different initiation steps. We propose the presence of an additional pathway (red arrows and text), thereby EAA limitation may directly lead to ribosomal stalling or delay during translation initiation (Met deficiency) and/or elongation (all EAA deficiencies), eventually resulting in epigenetic/transcriptional changes. The ERK and JNK branches of MAPKs are known to be activated during EAA starvation by yet unclear mechanisms.</p

GCN2 knockout does not interfere with transgene reactivation in HeLa cells.

<p>(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.</p

Gene set enrichment analysis of Met/Cys-deprived HeLa cells.

<p>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 <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200783#pone.0200783.s010" target="_blank">S1 File</a></b>. The enrichment analysis using all differentially expressed genes together did not reveal any additional enriched process.</p

EAA deprivation induces reactivation of silent transgenes in HeLa and HepG2 cells.

<p>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).</p