EMBO/SourceData huggingface dataset:
Figure 2D Genome browser view of an example DMR at a putative enhancer region. Group median CpG methylation is shown for no COPD (blue), COPD (I) (light green) and COPD (II-IV) (dark green). At the bottom the level of enhancer marks is depicted as fold-change over control: H3K4me1 (ENCODE accession: ENCFF102BGI) and H3K27ac (ENCODE accession: ENCFF386FDQ).H Enrichment of methylation sensitive transcription factor motifs at hypo- (right) and hypermethylated (left) DMRs. Methylation sensitive motifs were derived from the study Transcription factors, whose binding affinity was impaired upon methylation of their corresponding DNA motif are shown in red (MethylMinus) and transcription factors, whose binding affinity was increased, in blue (MethylPlus). TTS, transcription termination site. In H, Motif enrichment was calculated using HOMER, which uses ZOOPS scoring (zero or one occurrence per sequence) coupled with the hypergeometric enrichment calculations.
Figure 3D Selected examples of DEG across disease stages from clusters 2-5 defined in (C). In (D) each donor is represented by an individual point (no COPD n=3, COPD I n=3, COPD II-IV n=5), the group mean is shown as black dot and the error bar represents the standard deviation. In (D) FDRs and log2(fold-changes) (in A) were calculated using DESeq2, which uses a negative binominal GLM (generalized linear model) and Wald statistics. *: FDR <0.05; **: FDR <0.01; ***: FDR <0.001. The specific statistically significant FDR values are the following: RAD18 in COPD II-IV= 0.000434; ADAMTSL1 in COPD II-IV=0.02771; STEPA3 in COPD I= 0.04219 and in COPD II-IV= 0.001272; BMP4 in COPD II-IV= 0.0000535.
Figure 4F Scatter plots showing examples of correlations between gene expression and methylation of promoter associated DMRs. Each dot represents an individual donor. Dots are color coded according to disease state. Gene expression is illustrated as normalized counts. Methylation is illustrated as average beta value of the corresponding DMR. In (F) linear regression analysis was performed (black line) and the 95% confidence interval is indicated (grey area). Correlation coefficients and p-values were calculated by the Spearman correlation method.
Figure 5B Examples of primary pictures obtained in the screens showing the performance of the siRNA controls as well as positive hits upon KD. Scale bars: 0.05 mm. KD, knockdown; FGF, fibroblast growth factor; TGFβ, transforming growth factor beta; αSMA, alpha smooth-muscle actin; col1, collagen 1; NTC, non-targeting siRNA control;C Heatmap showing the effect of the KD of each candidate gene on the three measured readouts (αSMA, col1 and proliferation) in primary normal (NHLF) or COPD (DHLF) human lung fibroblasts, red: readout higher than NTC, blue: readout lower than NTC. |SSMD values| ≥ 2 are shown in lighter shade and |SSMD values| ≥ 3 in stronger shade. ; αSMA, alpha smooth-muscle actin; col1, collagen 1 SSMD, strictly standardized mean difference.
Figure 1(A) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA in Huh7 cells after 72 hours and 96 hours of infection. Data information: In A values were normalized to that of the GAPDH and represented as mean±SD of samples from five independent experiments. Student's t-test was applied to determine the significance between the groups.(B) Western blot analysis of the SARS-CoV-2 N protein (upper panel) and GAPDH (lower panel) levels in Huh7 cells, as mentioned in (A).(C-J) RT-qPCR analysis of the intracellular mRNA levels of HERV-T (C), HERV-E (D), HERV-R (E), HERV-V (F), HERV-FRD (G), HERV-MER34 (H), HERV-W (I) and HERV-K-HML-2 (J) in the SARS-CoV-2 infected Huh7 cells, as mentioned in (A). Data information: In C-J, values were normalized to that of the GAPDH and represented as mean±SD of samples from five independent experiments. Student's t-test was applied to determine the significance between the groups.
Figure 2(A) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA (normalized to that of the GAPDH) in Calu 3 cells after 48 hours and 72 hours of infection. Data information: In A values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.(B) Western blot analysis of the SARS-CoV-2 N protein (upper panel) and GAPDH (lower panel) levels in Calu 3 cells, as mentioned in (A).(C-J) RT-qPCR analysis of the intracellular mRNA levels of HERV-T (C), HERV-E (D), HERV-R (E), HERV-V (F), HERV-FRD (G), HERV-MER34 (H), HERV-W (I) and HERV-K-HML-2 (J) in the SARS-CoV-2 infected Calu 3 cells, as mentioned in (A). Data information: In C-J, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.
Figure 3(B) RT-qPCR measurement of exon 1 region [shown in (A)] transcript in Huh7 cells infected with SARS-CoV-2 for 72hours. (C) RT-qPCR measurement of exon 1 region transcript in Calu 3 cells infected with SARS-CoV-2 for 72hours. Data information: In B and C, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments.(E) RT-qPCR measurement of HERV-R-Env transcript level in PBMCs isolated from 19 healthy individuals and 16 COVID-19 patients. (F) RT-qPCR measurement of Exon1 region transcript level in PBMCs isolated from 19 Healthy individuals and 16 COVID-19 patients. Data information: values were normalized to that of the GAPDH and represented as mean±SD of samples from 19 healthy and 16 COVID-19 patients, assayed in duplicate.(G) Quantitation of HERV-R-Env RNA level in transcriptome dataset of indicated human lung tissues Data information: In G, HERV-R-Env copy number in two biological replicates (Healthy lung) and two technical replicates (COVID-19 lung) of the transcriptome dataset are represented as mean±SD.(H) Quantitation of HERV-R-Env RNA level in transcriptome dataset of Calu 3 cells, analyzed at 4, 8, 12 and 24 hours of SARS-CoV-2 infection, as indicated. Data information: In H, HERV-R-Env copy number in three biological replicates of the transcriptome dataset are represented as mean±SD. Student's t-test was applied to determine the significance between the groups.
Figure 4(A) RT-qPCR measurement of intracellular level of the SARS-CoV-2 delta variant RNA in Huh7 and Calu 3 cells after 24, 48 and 72 hours of infection. (B) RT-qPCR measurement of intracellular level of the SARS-CoV-2 omicron variant RNA in Huh7 and Calu 3 cells after 24, 48 and 72 hours of infection. Data information: values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.(C) RT-qPCR analysis of the HERV-R-Env RNA level in the SARS-CoV-2 delta variant infected Huh7 and Calu 3 cells, as mentioned in (A). (D) RT-qPCR analysis of the HERV-R-Env RNA level in the SARS-CoV-2 omicron variant infected Huh7 and Calu 3 cells, as mentioned in (B). Data information: values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.
Figure 5(B) Western blot analysis of the SARS-CoV-2 N protein (upper panel), HERV-R-Env protein (middle panel) and GAPDH (lower panel) levels in Huh7 cells, transiently transfected with HERV-R expression plasmid and infected with the SARS-CoV-2 for 24 and 48 hrs.(C) Densitometry quantification of N-protein band intensities (normalized to that of GAPDH) ) from three independent blots, as shown in Fig (B). Values are mean±SD of 3 independent experiments. Data information: In values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments.(F) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA (normalized to that of the GAPDH) in Huh7 cells transfected with NT-siRNA (Non target siRNA) or HERV-R-Env siRNA and infected with the SARS-CoV-2 for 24, 48 or 72 hrs, as indicated. Values are mean±SD of 3 independent experiments. Data information: In F, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments.(G) Dual luciferase assay showing activation of IFNγ, ISRE, ISG56 and NFκB promoters in HEK 293T cells expressing the empty vector (V), constitutively active mutant of RIG-I, R-C, 25 and 50 ng of HERV-R-Env. Activation of these promoters by R-C was taken as 100%. Data information: In G, FF Luc values were normalized to that of the RL Luc and represented as % mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.(H) Western blot analysis of the indicated proteins in Huh7 cells, transiently transfected with HERV-R-Env expression plasmid.
Figure 6(A) Western blot analysis of the indicated proteins in Huh7 cells, transiently transfected with HERV-R expression plasmid and infected with the SARS-CoV-2. (B) Densitometry quantification of P-ERK and N-protein band intensities (normalized to that of total-ERK and GAPDH, respectively) from three independent blots, representative image shown in panel (A). Data information: In B, P-ERK and N-protein band intensities were normalized to that of total-ERK and GAPDH, respectively [as shown in panel (A)] and represented as mean±SD of three independent blots. Student's t-test was applied to determine the significance between the groups.(C) Western blot analysis of the indicated proteins in Huh7 cells, transiently transfected with HERV-R expression plasmid, infected with the SARS-CoV-2 and treated with U0126. (D) Densitometry quantification of P-ERK and N-protein band intensities (normalized to that of total-ERK and GAPDH, respectively) from two independent blots, representative image shown in panel (C). Data information: In D, P-ERK and N-protein band intensities were normalized to that of GAPDH [as shown in panel (C)] and represented as mean of two independent blots. Individual values are shown as circles.
Figure 7(B) HERV-R 5'-LTR activity in Huh7 cells cotransfected with the pGL3 Fl 5'-LTR G-Luc and pGL3 promoter Firefly-Luc (denoted as Fl 5'-LTR G-Luc) or pGL3 G-Luc and pGL3 promoter Firefly-Luc (denoted as G-Luc) plasmids for 48 hours. (C) HERV-R 5'-LTR activity in Huh7 cells cotransfected with the pGL3 Fl 5'-LTR G-Luc and pGL3 promoter Firefly-Luc (denoted as Fl 5'-LTR G-Luc) or pGL3 ∆ 5'-LTR G-Luc and pGL3 promoter Firefly-Luc (denoted as ∆ 5'-LTR G-Luc) or pGL3 G-Luc and pGL3 promoter Firefly-Luc (denoted as G-Luc) plasmids for 48 hours. Data information: G-Luc values were normalized to that of the Firefly-Luc and represented as mean±SD of samples from three independent experiments.(I) RT-qPCR measurement of HERV-R-Env RNA in Huh7 cells infected with the SARS-CoV-2 and treated with the indicated compounds for 48 hours. Data information: In I values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.(J) Western blot analysis of the HERV-R-Env (upper panel), SARS-CoV-2 N (middle panel) and GAPDH (lower panel) protein levels in Huh7 cells infected with the SARS-CoV-2 and treated with the indicated compounds for 48 hours.(K) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA in Huh7 cells infected with the SARS-CoV-2 and treated with the indicated compounds for 48 hours. Data information: In K, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.
Figure 1B. BJ ESCs stably co-expressing H2B-GFP (green) and mCherry-H2B (red), hereafter named BJH2B-2FPs. C. ChIP-qPCR analysis of GFP enrichment inside different repetitive elements (ERV, MajSat, IAPs) used as H3K9me3 domain controls, or at the Hoxa11 gene used as a H3K27me3 domain control, or on transcribed genes (Gapdh, Pou5f1, Actb) or actively transcribed gene promoter (Pou5f1-promoter) in BJ WT and BJH2B-GFP ESCs. Gene names are color-coded in red whether they are associated to heterochromatic domains (enriched in H3K9me3); in black to Polycomb domains (enriched in H3K27me3); in blue to active chromatin domains (enriched in H3K4me3). Data are represented as relative enrichments of H2B-GFP versus histone H2B. Data are means ± s.d (n=3 biological replicates for GFP and histone H2B).E. Left panel, in vivo FLIM-FRET assay in BJH2B-2FPs stably co-expressing H2B-GFP (green) and mCherry-H2B (red). Mean FRET efficiency is displayed using a continuous pseudo-colour scale from 0 to 40%. Magnification of the FRET map, with discrete high FRET regions indicated by white arrowheads. Scale bars, 5 µm. Right panel, mean distribution of the FRET efficiency related to the pixel fraction from BJH2B-2FPs cells (n = 384 cells).
Figure 2A. Top and bottom panels depict a representative nucleus of BJH2B-2FPs ESC by GFP intensity (nucleosome concentration) and FRET efficiency, respectively. Yellow arrowheads: regions with high nucleosome density but low FRET efficiency; Orange arrowheads: regions with low nucleosome density but high FRET efficiency. Scale bars,10 μm. The right panel correlates FRET efficiency with GFP fluorescence intensity (Pearson correlation coefficient, r = -0.07, n = 106 cells).F. Top and bottom panels depict a representative nucleus of BJH2B-2FPs ESC after 15 min of ATP depletion by GFP intensity (i.e., nucleosome concentration) and FRET efficiency, respectively. Yellow arrows indicate nucleosome-rich foci associated with high levels of FRET. Scale bars,10 µm. The FRET efficiencies (%) from nucleosome-rich foci in untreated and ATP-depleted cells are depicted as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET efficiency (%) values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range. ****, p < 0.0001 (Mann-Whitney test, n = 61 cells). Mean distribution of the FRET efficiency of BJH2B-2FPs from nucleosome-rich foci in untreated (black, n = 162) and ATP-depleted cells (red, n = 61 cells). ****, p = 2.2e-16; K-S test.G. Left panel, representative image of in vivo FLIM-FRET measurements from differentiated 3T3 cells co-expressing H2B-GFP and mCherry-H2B. Yellow arrows indicate high FRET efficiencies at H2B-GFP bright chromocenters. Scale bars, 10 µm. Right panel, comparison of the FRET efficiency (%) from ESCs (blue) and 3T3 cells (white) in the GFP-brightest foci (nucleosome-rich foci). FRET efficiency (%) was represented as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range. ****, p < 0.0001 (Mann-Whitney test, n = 42 cells).
Figure 3A. Expression of mTagBFP-tagged HP1α in living BJH2B-2FPs and segmentation of HP1α positive foci. Yellow arrowheads: chromocenters enriched in H2B-GFP (left panel), in mTagBFP-HP1α (middle panel), and following segmentation (right panel). B. Box plot of the mean FRET efficiencies of all nucleosome-rich foci (black, n = 68 cells) and foci enriched in HP1α (blue, n = 68 cells). FRET efficiency (%) was represented as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the 10-90 percentiles value range. **** p < 0.0001, Mann-Whitney test.D. Representative image of the FRET efficiency map of interphase and mitotic ESCs (top and bottom panels, respectively) after fixation with 4% PFA. The mean FRET efficiency is displayed using a continuous pseudo-colour scale from 0 to 80%.E. Mean distribution of FRET efficiency from nucleosome-rich foci in living cells (green, n = 162 cells), and fixed cells (black, n = 157 cells). ****, p = 2.2e-16; K-S test. Right panel, box plot of the mean FRET efficiency from nucleosome-rich foci in living and fixed ESCs. FRET efficiency (%) was represented as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range. **** p < 0.0001, Mann-Whitney test.
Figure 4A. Total cell extracts from BJH2B-2FPs cells incubated during 24h with untargeted siRNA or an siRNA targeting HP1α, analyzed by western blotting with an antiserum against HP1α. Loading control was performed by red ponceau staining.C. Left panel, mean distribution of the FRET efficiency from nucleosome-rich foci of BJH2B-2FPs cells incubated with a control siRNA (black, n = 101 cells) or an siRNA against HP1β (green, n = 136 cells); *, p: 0.02651; K-S test. Right panel, box plot of the mean FRET efficiency from nucleosome-rich foci of living of BJH2B-2FPs cells incubated with a control siRNA (grey, n = 101 cells) or an siRNA against HP1β (green, n = 136 cells). Box-and-Whisker plots represent the FRET efficiency (%). The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range.E. Left panel, representative images of immunostaining for H4K20me3 in control ESCs (top row) and ESCs treated with A-196 during 2 days (bottom row). Scale bars, 10 µm. Right panel, Box-and-Whisker plots represent the normalized fluorescence intensity of immunostaining for H4K20me3. The box plots indicate median values (middle lines), mean values (middle crosses), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the fluorescence intensity. Data are means of n=2 biological replicates. Number of nuclei = 76/60 for control ESCs/A-196 treated ESCs.F. Total cell extracts from untreated, DMSO treated or A-196 treated BJH2B-2FPs during 24h or 48h, analysed by western blotting with antibody against H4K20me3. Arrowhead indicates the H4K20me3-specific bands. The asterisk indicates unspecific bands.
Figure 5A. Representative images of immunostainings for Ki-67 in mitotic WT, control (CTRL) and Mki67-/- ESCs. Scale bar, 10 µm.B. Box-and-Whisker plots represent the FRET efficiency (%) from control mitotic ESCs (n = 12 cells) and from three different Mki67-/- ESCs clones named #1 (n = 10 cells), #2 (n = 9 cells) and #3 (n = 8 cells). The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the 10-90 percentiles value range; ***, p = 0.0007; ****, p < 0.0001, Mann-Whitney test.C. Left panel, in vivo FLIM-FRET measurements in interphase BJH2B-2FPs control ESCs (top) and BJH2B-2FPs Mki67-/- ESCs (clone #1). Mean FRET efficiency is displayed using a continuous pseudo-colour scale from 0 to 25%. Scale bars, 10 µm. Right panel, box-and-Whisker plots represent the FRET efficiency (%) in WT ESCs (n = 77 cells) and Mki67-/- ESCs clones clone #1 (n = 86 cells), #2 (n = 95 cells) and #3 (n = 110 cells). The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the 10-90 percentiles value range. ****, p < 0.0001, Mann-Whitney test.
Figure 6B. Left panels, nucleosome-rich foci segmentation in naive ESCs and EpiLCs using the FRENETIC tool. Nuclei are outlined with yellow dashed lines. Scale bars, 10µm. Right panel, Box-and-Whisker plots of the mean number of foci per nucleus in naive BJH2B-2FPs ESCs (n = 150 cells) and in primed EpiLCs (n = 137 cells). The thick line represents median, the boxes correspond to the number of foci from the 25-75th percentiles of the medianC. Left panel, Box-and-Whisker plots represent the FRET efficiencies (%) from naive ESCs (blue, n = 162 cells) and EpiLCs (red, n = 266 cells). The box plots indicate median values (middle lines), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the FRET efficiency; ns, p = 0.29, Mann-Whitney test. Right panel, mean distribution of the FRET efficiency related to the pixel fraction in nucleosome-rich foci from naive ESCs (blue, n = 162 cells) and EpiLCs (red, n = 266 cells).D. Mean distribution of the FRET efficiency related to the pixel fraction in nucleosome-rich foci from EpiLCs at different time points of cellular induction (black, +24h n= 57 cells; orange, +48h, n= 48 cells; red, +72h n = 266 cells). ****, p = 2.2e-16; K-S test.E. Total cell extracts from naive ESCs and EpiLCs (+72h) analyzed by western blot with antiserum against H4K20me3. Short and long exposures of the signal are shown. Loading control was achieved by Ponceau staining.F. Left panel, mean distribution of the FRET efficiency related to the pixel fraction in nucleosome-rich foci from untreated EpiLCs (black, n = 124 cells) and A-196 EpiLCs treated cells (red, n = 113 cells). ****, p = 2.2e-16; K-S test. Right panel, Box-and-Whisker plots indicate the FRET efficiency (%) from untreated EpiLCs (black, n = 124 cells) and treated with A-196 during 2 days (red, n = 113 cells). The box plots indicate median values (middle lines), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the FRET efficiency.
Figure 1NIX and BNIP3 are required for mitophagy induced by MLN4924 and other HIF1α stabilisers. Parental HeLa cells and NIX/BNIP3 double knockout (NIX/BNIP3 DKO) HeLa cells expressing mt-Keima were treated with DFP (1 mM), MLN4924 (0.5 μM), or DMOG (10 nM) for 24 hours and analysed by live-cell confocal microscopy. The emission signals obtained after excitation with the 458 nm laser (neutral pH) or 561 nm laser (acidic pH) are shown in green and magenta, respectively. Quantification of mitophagy shown in panel A. Mitophagy is represented as the ratio of mt-Keima 561 nm fluorescence intensity to mt-Keima 458 nm fluorescence intensity for individual cells normalised to the mean of the untreated condition. In B , the measurements from individual cells are represented as translucent grey dots, and the mean ratio from each independent experiment is represented by coloured circles. The centre lines and bars depict the mean of the averaged independent replicates +/- standard deviation. N=4. A minimum of 50 cells were analysed for each condition within each individual replicate experiment, and over 300 cells were analysed for each condition in total. P values were calculated based on the mean ratio values from independent experiments using one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001).Analysis of NIX and BNIP3 protein levels after MLN4924, DMOG, or DFP treatments. HeLa cells or HeLa NIX/BNIP3 DKO cells were treated with MLN4924, DMOG, or DFP for 24 hours. Total-cell lysates corresponding to conditions in panel A were subject to immunoblotting.Inhibition of HIF1α with echinomycin prevents DFP-induced mitophagy but not MLN4924-induced mitophagy. U2OS cells expressing mt-Keima were treated with the indicated drugs for 24 hours and analysed by live-cell confocal microscopy. Quantification of mitophagy shown in panel D. In F, the measurements from individual cells are represented as translucent grey dots, and the mean ratio from each independent experiment is represented by coloured circles. The centre lines and bars depict the mean of the averaged independent replicates +/- standard deviation. N=4. A minimum of 50 cells were analysed for each condition within each individual replicate experiment, and over 300 cells were analysed for each condition in total. P values were calculated based on the mean ratio values from independent experiments using one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001).Inhibition of HIF1α with echinomycin prevents the increase of NIX and BNIP3 in response to DFP, but not MLN4924. U2OS cells were treated with DFP or MLN4924 over a time course. Where indicated, cells were also treated for 24 hours with echinomycin. Total-cell lysates were subject to immunoblotting as shown.