EMBO/soda-nel huggingface dataset:
Expression of endogenous retroviruses reflects increased usage of atypical enhancers in T cells (2019)
Figure 3

Figure 3: Dieldrin causes transcription of HERV-containing PEs atypical for activated T cells

Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs.

(A) Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin. Top tracks show coverage from sequencing of poly(A)-enriched libraries for comparison. All coverage tracks are at the same scale and all the sequencing result files are of comparable size. Blue and red reads (rds) are in opposite orientation. CAGE peaks use the same color code.

(B) Reads mapping inside a series of 4686 HERVs located away (30Kb) from protein-coding genes were quantified both in the poly(A)-enriched and the ribo-depleted data. Read counts from each replicate were then summed and plotted.

(C) Distribution of regions annotated "LTR" in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO.

(D) Schematic showing that only a fraction of the APEs (sites of divergent transcription activated 2-fold or more by dieldrin) match promoters and enhancers annotated as active in T cells (APETs). The remaining APEs are designated APEDs (specific to the dieldrin treatment).

(E) Comparison of regions annotated "LTR" in RepeatMasker with the APEs, APETs, and APEDs. The graph provides the ratio between the Jaccard index of the indicated category over the average of Jaccard indexes calculated for 1000 series of randomly selected PEs.

(F, G) Comparison of either APEs or APEDs as indicated with the regions annotated TssA, TssAFlnk, Enh, EnhG, TssBiv, or EnhBiv in the 15 core marks model of the Epigenomic Roadmap consortium as in Figure 1A.

(H) Region upstream of IL2 as a screen capture from the genome browser of the Epigenomic Roadmap consortium. Promoters and enhancers are annotated in red and yellow respectively. Green indicates transcription. Insert is a detail from Appendix Figure S3A showing the site of divergent transcription induced by dieldrin.

(I) Jurkat T cells were treated with either DMSO (vehicle) or 100μM dieldrin for the indicated times. Abundance of IL2 mRNA was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001.

(J) Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22. Color code as in (A).
Expression of endogenous retroviruses reflects increased usage of atypical enhancers in T cells (2019)
Figure 4

Figure 4: Dieldrin activates multiple signal transduction pathways.

(A) Schematic of the signal transduction pathways under scrutiny. Used inhibitors are indicated in bold font.

(B) A human phospho-kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO, dieldrin, or PMA for 30 min as indicated. The array processed as a western and revealed by ECL. Graphic shows selected antibodies. Values shown are means +/- SEM from two measures. The full experiment is shown in Appendix Figure S4A and S4B.

(C) Jurkat cells were treated with either DMSO (vehicle), dieldrin, or PMA for the indicated times. Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies. Nearest size marker is indicated to the right (kDa).

(D-G) Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001, **: 0.001<p-value<0.01, *: 0.01<p-value<0.05.

(H) Jurkat cells treated as in (C) were subject to fractionation as indicated in the Material and Method section to distinguish a fraction with mostly RNAPII S2p (F1), one with mostly histone H3 (F3), and one with both RNAPII S2p and histone H3 (P). Fractions were resolved by PAGE, and western blots were carried out with the indicated antibodies.
Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria (2021)
Figure 1

Figure 1: EPODs are highly robust across growth conditions.

(A) Example regions exhibiting the IPOD-HR occupancy where EPOD calls are made. Loose EPODs (pink) and Strict EPODs (teal) represent spans of high protein occupancy at lower and higher thresholds, respectively. Neighboring areas showing lower robust z scores are not EPODs. Note that following the definitions in [12], the presence of an EPOD requires a sustained region of high average occupancy, without any regions in which occupancy drops to background levels. Thus, breaks in EPOD calls represent cases where a rolling average occupancy drops below the calling threshold (e.g., the breakpoint in the middle of pinQ in the top panel), and regions of apparent occupancy may nevertheless not be called due to being below the minimum length threshold (as is the case for nohA).

(B) Strict EPOD counts across WT (MG1655) E. coli cells are similar across different conditions. Cells were grown in: Rich (Rich Defined Media (See Materials and Methods)), Min (Minimal Media) and collected at log phase growth or deep stationary phase (Deep Stat.). WT (2) is a MG1655 isolate from another laboratory that differs from our WT strain by four-point mutations and two insertion sequence integrations.

(C) Fractional coverage of Strict EPODs across the genome does not largely vary across conditions, however cells grown in the Min condition have a slightly larger coverage.

(D) To assess the similarity between EPOD calls in each condition, we calculated the symmetrized overlap distance of EPODs. A value of 0 indicates that the set of EPODs are identical. Hierarchical clustering reveals that growth phase impacts EPOD location in this dataset.

(E) Specific locations across all conditions remain occupied by protein where EPODs across a representative region, the waa operon, were called (left) and unoccupied by RNA polymerase (right). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy.

(F) Kernel density plots displaying the normalized histograms (smoothed by a kernel density estimator) of H-NS ChIP [20] for regions of the genome within EPODs versus Background (i.e., protein occupancy through the rest of the genome, where contiguous regions between EPODs were treated as a single data point). H-NS binding shows a strong overlap with EPOD locations measured in all conditions. (*) indicates FDR-corrected p<0.005 via permutation test indicating an enrichment in H-NS ChIP binding in EPODs across conditions (against a null hypothesis of no difference in medians).
Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria (2021)
Figure 2

Figure 2: Loss of nucleoid associated proteins (NAPs) leads to changes in EPODs. (A) Number of EPODs called in different NAP deletions. D.S. denotes genotypes where cells were collected in the deep stationary phase of growth. (B) Fractional coverage of EPODs for each genotype across the genome. (C) Symmetrized overlap statistic comparing each pair of samples in the NAP deletion dataset. The symmetrized overlap denotes similarity between EPOD locations, where a value of 0 = identical. Hierarchical clustering was performed to group like-genotypes. (D) Distributions of mean protein occupancy at WT EPOD boundaries and background. The blue dots denote the median and the black line displays the interquartile ranges in each condition. The dashed pink line represents the WT median. (*) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs. WT for each condition that has been adjusted using the Benjamini and Hochberg method (against a null hypothesis of no difference in pseudomedians). The grey line denotes the same comparison between the D.S. conditions. Underlined (*)'s indicate a gain in the pseudomedian compared to baseline conditions, while no underline indicates a loss in the pseudomedian compared to baseline conditions. p value < 0.05 = *, <0.005=**, <0.0005=***. Three biological replicates were used for the "WT, Rich" case and "WT (deep stat.)" case, and two biological replicates for the "WT, Min" and "WT (2), Rich" conditions. (E) Protein occupancy over the waa operon. The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. The EPOD over the waa operon is lost in the ∆stpA∆hns condition which results in increased accessibility and RNA polymerase occupancy (Fig. S3A). (F) The loss in protein occupancy shown in Figure 2E leads to increases in RNA polymerase occupancy across the waa operon. (G) RNA-seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA. (*) indicate q value < 0.05 = *, < 0.005 = **, < 0.0005 = *** (called using DeSeq2, as described in Methods).
Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria (2021)
Figure 3

Figure 3: Changes in EPODs are induced in specific conditions.

(A) Experimental overview. WT, MG1655 cells were grown in M9 Minimal Media with 0.2% glucose, samples were collected at mid log phase of growth (OD600 ~0.2) for RNA-seq and IPOD-HR. The cells were back diluted to an OD600 of ~0.1 in M9 Minimal Media with 0.2% 5-Keto-D-gluconic-acid (5KDG), grown to an OD600 of ~0.2, collected for RNA-seq and IPOD-HR. In the final shift, the cells were back diluted to an OD600 of ~0.003 in M9 Minimal Media with 0.2% glucose, grown to OD600 of 0.2 and collected for RNA-seq and IPOD-HR. Two biological replicates were performed.

(B) Protein occupancy over the idn operon for each condition (colors are denoted in (A)). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. There is a large loss in protein occupancy when cells are shifted to 5KDG, leading to the loss of the call

ed EPOD. Protein occupancy is restored once cells are grown in the second glucose condition. The 500bp normalized average of previously published H-NS ChIP-seq [20] exhibits high H-NS binding on the idnD promoter region.

(C) To examine the expression of the idn operon at each shift, RNA-seq was performed. RNA-seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition (as colored in (A)). Comparisons are denoted with colored dots with significance stars representing the adjusted p-value where (***) signify q-values <0.0005 (called using DeSeq2, as described in Methods).

(D) Spearman correlations are represented with the heatmap comparing the expression profiles in each condition, where identical expression values for every gene show a spearman correlation of 1. The symmetrized overlap distance was calculated for all EPODs for each condition, where a value of 0 is identical. The colored squares on the sides of the heatmaps denote the condition (following the colors shown in (A)).
RASSF1C oncogene elicits amoeboid invasion, cancer stemness and extracellular vesicle release via a SRC/Rho axis (2021)
Figure 1

Figure 1 The RASSF1C oncogene promotes mesenchymal-amoeboid transition

A. Top: graphs showing the change in cell diameter for MCF7 cells transfected with ZsGreen (left) or ZsRASSF1C (right) plasmids at 0 h and 12 h, connections indicate individual cell tracking. Bottom: example images diameter measurements (red line). Scale 20 μm.

B. Single cell morphology of MCF7 and MDA-MB-231 cell lines, expressing or not a RASSF1C construct, cultured in 3D rat tail collagen I and analyzed for their rounded or elongated morphology 24 hours after seeding in 3D matrix.

C. Confocal images of MDA-MB-231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D-collagen matrix. Cells were imaged and stained with Phalloidin-568 (red), pMLCII/Alexa‑633 (false‑coloured green) or imaged for ZsGreen (false-coloured white). Scale 20 μm.

D. Single cell morphology assay of MDA-MB-231 cells cultured in 3D-collagen and analysed for morphology 24 hours after transient knock-down using either a control sequence (NT, non-targeting) or a sequence targeting the RASSF1 locus. RASSF1 knock-down marks a reduction in rounded cells.

E. Gelatine zymography assay shows downregulation of MMP-9 metalloprotease in MDA-MB-231 cells over‑expressing RASSF1C.

F. 3D Matrigel Boyden chamber invasion with or without metalloproteases inhibitor GM6001 and

3D Hydrogel invasion of MDA-MB-231 control cells or cells over‑expressing RASSF1C (24 h).

G. Quantification of Boyden chamber invasion after 24 hours in 3D matrigel of MDA-MB-231 cells, transfected with ZsGreen (control) or ZsRASSF1C (RASSF1C) and treated with inhibitors against ROCK (Y27632, 10 μmol/L) or Rho (C3, 2 μg/ml).

H. Cartoon summarising intravital imaging experiments.

I. Lung colonization of MDA-MB-231 CFP+ cells (MDA-MB-231CFP) stably expressing control or HA-RASSF1C in mammary gland tumors from 5 mice each, measured from intravital imaging time-lapse images.

J. Average migration speed (expressed as μm/min) of n=30 randomly selected CFP positive cells in intravital images of Control and RASSF1C expressing MDA-MB-231 tumors.

K. Quantification of CFP+ MDA-MB-231 cells displaying different types of morphology in vivo, expressed as average number of events, observed from a total of 30 positions in n=5 mice/group (Control or RASSF1C). Mesenchymal, amoeboid and hybrid morphology were scored manually based on diameter and circularity (distance between the two furthest points). The higher distance between cell edges was defined as mesenchymal morphology, while the lower distance was considered amoeboid morphology.

Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
RASSF1C oncogene elicits amoeboid invasion, cancer stemness and extracellular vesicle release via a SRC/Rho axis (2021)
Figure 2

Figure 2 RASSF1C oncogene upregulates Rho/ROCKI/pMLCII signaling pathway via SRC

A. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 and MCF7 cell lines transiently transfected either with control or Flag-RASSF1C plasmid.

B. Rho-GTP pull-down binding assay in MDA-MB-231 control or RASSF1C over-expressing cells and co‑transfected either with SRC plasmids, siRNA against SRC or siNT (non-targeting sequence). Western blot analysis shows Rho activity (Rho GTP-double band) and pMCLII and pRhoGDI binding. The ratio below Rho-GTP Western blot shows differences in Rho-GTP protein expression, compared to control condition.

C. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control.

D. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA‑RASSF1C plasmid. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies.

E. Schematic cartoon recapitulating the proposed RASSF1C/SRC‑driven mechanism.

F. Top, schematic of RASSF1C domain structure indicating putative mutations in the RA domain affecting RhoA activity. Bottom, immunoprecipitation of MDA-MB-231 transiently transfected with either control (DsRed), DsRASSF1C plasmid, DsRASSF1C-R197W or DsRASSF1C-R199F mutants. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies.

G. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 cells transiently transfected with RASSF1C or its mutants (R197W, R199F). The ratio under Rho-GTP Western blot shows differences in active Rho protein expression, compared to control condition.

H. Quantification of rounded versus elongated cells in single cell morphology assay in 3D‑collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed.

I. Representative confocal images show elongated or rounded cells per field of view of MDA‑MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D‑collagen and stained with Phalloidin-568 (red) for F‑actin and pMLCII/Alexa 633. Scale bars represent 10 μm.

Data information: For both Rho-GTP pull-downs and immunoprecipitation assays total proteins were used as loading controls. All data are from n=3 independent experiments. Data are analyzed by Student T-test, represented as mean ± SEM. *** p ≤ 0.001.
RASSF1C oncogene elicits amoeboid invasion, cancer stemness and extracellular vesicle release via a SRC/Rho axis (2021)
Figure 3

Figure 3 Amoeboid RASSF1C cells produce pro-invasive Extracellular Vesicles

A. Left: Cartoon showing the principle behind the Cre-carrying EVs and how recipient cells switch from DsRed+ to eGFP+ expression upon Cre-mediated excision of the DsRed locus. Centre: representative images, and right: quantification of T47DDsRed cells that converted to eGFP+ in the bottom wells of a transwell system in the presence of MDA-MB-231CFP;Cre;Control or MDA‑MB‑231CFP;Cre;HA-RASSF1C seeded in the upper wells. Scale bars represent 100 μm.

B. Left: averaged Nanoparticle Tracking Analysis displaying the size distribution of EVs isolated from media of MDA-MB-231 cells expressing Control (pcDNA3) or FLAG-RASSF1C plasmid. Inserts: transmission electron microscopy images of single EVs. Scale bars represent 200 nm. Right: quantification of total secreted EVs (96 h, top) or at indicated times (below), as measured by NTA.

C. Western blot analysis of EVs isolated from MDA-MB-231 cells transfected with Control (pcDNA3) or FLAG-RASSF1C with indicated antibodies. Equal amounts of protein were loaded after normalization via microBCA assay.

D. Quantification of EVs isolated from MDA-MB-231 cells transfected with Control (pcDNA3), RhoA‑CA, RhoA‑DN, FLAG‑RASSF1C, FLAG-RASSF1C‑R199F, or FLAG‑RASSF1C + RhoA‑DN using Nanoparticle Tracking Analysis.

E. Normalized values of real‑time migration rates in xCELLigence plates of MDA-MB-231 cells treated with 20 ng/μl EVs derived from MCF7 cells expressing a Control, FLAG-RASSF1C, or EGFP-RhoA plasmid, left to migrate for 24 h. Statistics were performed with 2-way ANOVA (Dunnett's multiple comparisons test).

F. Left: Representative images of Boyden chamber invasion assay in 3D matrigel of MCF7 cells, treated with 20 ng/μl EVs derived from MDA-MB-231 cells, transfected the same constructs used in (e). Right: Quantification of Boyden chamber invasion 24 hours after EV treatment. Scale bars represent 200µm.

G. Top: graphical representation of the functional (invasion) assay performed using MDA‑MB‑231 derived EVs on MCF7 recipient cells. Bottom: Boyden chamber invasion assay in 3D matrigel of EVs isolated from different steps of ultracentrifugation. EVs were collected from conditional medium produced by MDA-MB-231 expressing Control (pcDNA3) or FLAG-RASSF1C. The 2K (large EVs), 10K (medium EVs or MVs) and 100K (small EVs, including exosomes) pellets were collected and equal amount of EVs (normalized according to protein content) were incubated with MCF7 cells. MCF7 were evaluated for invasion after 24 hours.

Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
RASSF1C oncogene elicits amoeboid invasion, cancer stemness and extracellular vesicle release via a SRC/Rho axis (2021)
Figure 4

Figure 4 Amoeboid RASSF1C cells express cancer stemness features

A. Quantification of fluorescent intensity of CD133 and ALDH1 cancer stem cell markers in MDA‑MB‑231 control or RASSF1C transfected spheroids cultured on Matrigel matrix.

B. Confocal images of ZsGreen or ZsRASSF1C expressing MDA-MB-231 spheroids embedded in 3D rat tail collagen I. Cells were stained with NANOG (red). Zoom images show that RASSF1C expressing cells are also NANOG positive. Scale 100 μm.

C. Western blot analysis of CD133 protein expression level in MDA-MB-231 cells treated with siNT or siRASSF1.

D. Representative confocal images (upper row) and quantification of fluorescent intensity of ALDH1 expression in MDA-MB-231 cells expressing RASSF1C (black bar in the graph) or after siRNA treatment against RASSF1 (grey bar). Lower row shows merged images with DAPI (blue), ALDH1 (green) and RASSF1C (red). Scale bars represent 20 μm.

E. Quantification of the absolute number of NANOG positive cells (measured as average cell number/spheroid) per MDA-MB-231 ZsGreen (control) or ZsRASSF1C (RASSF1C) spheroids in 3D-collagen, treated with ROCK inhibitor (Y27632, 10 μmol/L) or Rho inhibitor (C3, 2 μg/ml).

F. Quantification of the absolute number of NANOG positive cells (measured as average cell number/spheroid) per MDA-MB-231 ZsGreen (control, con) or ZsRASSF1C (RASSF1C) spheroids in 3D-collagen, co‑transfected with either RhoA WT, catalytically active (CA) or dominant negative (DN) constructs.

G. Fold change in qRT-PCR of NANOG, OCT4 and SOX2 mRNA from MCF7 cells expressing CRISPR‑dCas9 or dCas9‑VP64 plus single guide RNA for the RASSF1C promoter (sgRASSF1C).

H. Quantification of the percentage of double positive pMLCII and NANOG cells in MCF7 cells expressing CRISPR-dCas9, dCas9-VP64, dCas9‑SKD plus single guide RNA for the RASSF1C promoter (sgRASSF1C). At least 300 cells were counted per experiment.

I. Quantification of number of invaded MCF7 cells in 3D matrigel, after incubation with EVs from MDA-MB-231 cells sorted for Greenhigh expression. MDA-MB-231 cells stably expressing a pQXCIP‑zsGreen or pQXCIP‑zsGreen-RASSF1C construct were sorted and EVs were harvested 24 h later, prior to seeding with MCF7 cells in Boyden chambers. Invasive potential of MCF7 cells was evaluated 24 h after incubation with EVs.

J. qRT‑PCR expression of NANOG, OCT4, SOX2 in MCF7 cells 24 h after exposure to 50 ng/μl EVs derived from MDA‑MB‑231 cells (treated as indicated).

Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.